Rapid clearance of antigen complexes using novel antibodies

ABSTRACT

The present invention relates to rapid clearance molecules that bind target antigens and FcγRIIb with increased affinity as compared to parent molecules, the compositions being capable of causing accelerated clearance of such antigens. Such compositions are useful for treating a variety of disorders, including allergic diseases, atherosclerosis, and a variety of other conditions.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application Ser.Nos. 61/752,955, filed Jan. 15, 2013; 61/794,164, filed Mar. 15, 2013,61/794,386, filed Mar. 15, 2013, and 61/833,696, filed Jun. 11, 2013,each of which is expressly incorporated by reference in the entirety.

RELATED APPLICATIONS

U.S. Ser. Nos. 11/124,620, 13/294,103, 12/341,769 and 12/156,183 are allexpressly incorporated by reference in their entirety, particularly forthe recitation of amino acid positions and substitutions, and all data,figures and legends relating thereto.

TECHNICAL FIELD

The present disclosure relates to methods of using polypeptides with twodomains, a first domain that bind a ligand (such as the variable regionof an immunoglobulin or a fusion partner) and a second domain, an Fcdomain, that binds FcγRIIb, particularly human FcγRIIb, with highaffinity. These methods resulting in rapid and accelerated clearance ofthe polypeptide-ligand complexes, e.g. the antibody-antigen complexes inthe case of antibody polypeptides. Such methods are useful for treatinga variety of conditions.

BACKGROUND OF THE INVENTION

Antigen recognition by B cells is mediated by the B cell receptor (BCR),a surface-bound immunoglobulin in complex with signaling componentsCD79a (Igα) and CD79b (10). Crosslinking of BCR upon engagement ofantigen results in phosphorylation of immunoreceptor tyrosine-basedactivation motifs (ITAMs) within CD79a and CD79b, initiating a cascadeof intracellular signaling events that recruit downstream molecules tothe membrane and stimulate calcium mobilization. This leads to theinduction of diverse B cell responses (e.g., cell survival,proliferation, antibody production, antigen presentation,differentiation, etc.) which lead to a humoral immune response(DeFranco, A. L., 1997, Curr. Opin. Immunol. 9, 296-308; Pierce, S. K.,2002, Nat. Rev. Immunol. 2, 96-105; Ravetch, J. V. & Lanier, L. L.,2000, Science 290, 84-89). Other components of the BCR coreceptorcomplex enhance (e.g., CD19, CD21, and CD81) or suppress (e.g., CD22 andCD72) BCR activation signals (Doody, G. M. et al., 1996, Curr. Opin.Immunol. 8, 378-382; L1, D. H. et al., 2006, J. Immunol. 176,5321-5328). In this way, the immune system maintains multiple BCRregulatory mechanisms to ensure that B cell responses are tightlycontrolled.

When antibodies are produced to an antigen, the circulating level ofimmune complexes (e.g., antigen bound to antibody) increases. Theseimmune complexes downregulate antigen-induced B cell activation. It isbelieved that these immune complexes downregulate antigen-induced B cellactivation by coengaging cognate BCR with the low-affinity inhibitoryreceptor FcγRIIb, the only IgG receptor on B cells (Heyman, B., 2003,Immunol. Lett. 88, 157-161). It is also believed that this negativefeedback of antibody production requires interaction of the antibody Fcdomain with FcγRIIb since immune complexes containing F(ab′)₂ antibodyfragments are not inhibitory (Chan, P. L. & Sinclair, N. R., 1973,Immunology 24, 289-301). The intracellular immunoreceptor tyrosine-basedinhibitory motif (ITIM) of FcγRIIb is necessary to inhibit BCR-inducedintracellular signals (Amigorena, S. et al., 1992, Science 256,1808-1812; Muta, T., et al., 1994, Nature 368, 70-73). This inhibitoryeffect occurs through phosphorylation of the FcγRIIb ITIM, whichrecruits SH2-containing inositol polyphosphate 5-phosphatase (SHIP) toneutralize ITAM-induced intracellular calcium mobilization (Kiener, P.A., et al., 1997, J. Biol. Chem. 272, 3838-3844; Ono, M., et al., 1996,Nature 383, 263-266; Ravetch, J. V. & Lanier, L. L., 2000, Science 290,84-89). In addition, FcγRIIb-mediated SHIP phosphorylation inhibits thedownstream Ras-MAPK proliferation pathway (Tridandapani, S. et al.,1998, Immunol. 35, 1135-1146).

A recently recognized function of FcγRIIb is to serve as a scavengerreceptor in the liver, clearing antibody:antigen immune complexes fromcirculation. FcγRIIb is thus an important component of the classicalreticulo-endothelial system. For example, Anderson and colleagues(Ganesan et al., J Immunol 2012) published a study demonstrating thatthree quarters of mouse FcγRIIb is expressed in the liver, with 90% ofit being expressed in Liver Sinusoidal Endothelial Cells (LSEC).Moreover, the authors demonstrated that clearance of radiolabeled smallimmune complexes (SIC) is significantly impaired in an FcγRIIb knockoutstrain compared to wild-type mice. This is therefore a natural propertyof the immune system, which can be accentuated by Fc engineering forenhanced affinity to FcγRIIb.

Of relevance in the present invention are allergic diseases. Allergicdiseases and conditions, such as asthma, allergic rhinitis, atopicdermatitis, and food allergy, have become increasingly prevalent overthe past few decades and now affect 10-40% of the population inindustrialized countries. Allergic diseases profoundly affect thequality of life, and can result in serious complications, includingdeath, as may occur in serious cases of asthma and anaphylaxis.Allergies are prevalent, and are the largest cause of time lost fromwork and school and their impact on personal lives as well as theirdirect and indirect costs to the medical systems and economy areenormous. For example, allergic rhinitis (hay fever) affects 22% or moreof the population of the USA, whereas allergic asthma is thought toaffect at least 20 million residents of the USA. The economic impact ofallergic diseases in the United States, including health care costs andlost productivity, has been estimated to amount to $6.4 billion in theearly nineties alone.

Most allergic diseases are caused by immunoglobulin E (IgE)-mediatedhypersensitivity reactions. IgE is a class of antibody normally presentin the serum at minute concentrations. It is produced by IgE-secretingplasma cells that express the antibody on their surface at a certainstage of their maturation. Allergic patients produce elevated levels ofIgE with binding specificity for ordinarily innocuous antigens to whichthey are sensitive. These IgE molecules circulate in the blood and bindto IgE-specific receptors on the surface of basophils in the circulationand mast cells along mucosal linings and underneath the skin. Binding ofantigen or allergen to IgE on mast cells, basophils, and other celltypes, crosslink the IgE molecules, and aggregate the underlyingreceptors, thus triggering the cells to release vasoactive and neuronalstimulatory mediators such as histamines, leukotrienes, prostaglandins,brakykinin, and platelet-activating factor. The rapid reaction of theimmune system to antigen caused by antibody immune complexes has led tothe term immediate or antibody-mediated hypersensitivity reaction, incontrast to delayed or cell-mediated hypersensitivity reactions that aremediated by T cells. IgE-mediated immune reactions are specificallyreferred to as type I hypersensitivity reactions.

The high affinity receptor for IgE (FcεRI) is a key mediator forimmediate allergic manifestations. In addition to mast cells andbasophils, the primary mediators of allergic reactions, FcεRI is foundon a number of other cell types including eosinophils, platelets and onantigen-presenting cells such as monocytes and dendritic cells. Anadditional receptor for IgE is FcεRII, also known as CD23 or thelow-affinity IgE Fc receptor. FcεRII is expressed broadly on Blymphocytes, macrophages, platelets, and many other cell types such asairway smooth muscle. FcεRII may play a role in the feedback regulationof IgE expression and subsequently FcεRII surface expression.

Since IgE plays a central role in mediating most allergic reactions,devising treatments to control IgE levels in the body and regulating IgEsynthesis has been of great interest. Several strategies have beenproposed to treat IgE-mediated allergic diseases by downregulating IgElevels. One strategy involves neutralizing the IgE molecules by bindingthe ε-chain of IgE in or near the Fc-receptor binding site. For example,Omalizumab (Xolair) is a recombinant humanized monoclonal anti-IgEantibody that binds to IgE on the same Fc site as FcεRI. Omalizumabcauses a reduction in total serum or circulating IgE in atopic patients,which attenuates the amount of antigen-specific IgE that can bind to andsensitize tissue mast cells and basophils. This, in turn, leads to adecrease in symptoms of allergic diseases. Interestingly, serum IgElevels increase after start of therapy because of omalizumab-IgE complexformation and may remain high up to a year after stopping therapy.Consequently, this issue may lead to false-negatives on diagnostic testsand therefore IgE levels must be routinely checked. Accordingly, thereexists a need for improved methods and compositions to reduceIgE-mediated diseases and disease symptoms.

Of additional relevance in the present invention is the fact thatantibody/antigen immune complexes are well established mediators ofinflammation in various autoimmune diseases. Moreover, circulatingimmune complexes can be deposited in the kidney, ultimately resulting innephritis, the leading cause of death in systemic lupus erythematosus(SLE). Finally, nucleic-acid (RNA or DNA) containing immune complexes,observed most notably in SLE, can interact with toll-like receptors(TLRs) on immune cells, inducing the release of inflammatory cytokinessuch as interferon alpha, contributing to disease pathogenesis. Thecomplement system naturally recognizes these antibody-antigen immunecomplexes (ICs), resulting in complement-component C3 ‘tagging’ of theimmune complexes with a variety of fragments of C3 (including C3b,C3b(i), C3d, and C3g). Under healthy conditions, these tagged immunecomplexes are cleared through interaction with a variety of complementreceptors and FcγRs. C3b-C3b-IgG covalent complexes are immediatelyformed on interaction of serum C3 with IgG-IC. These C3b-C3b dimersconstitute the core for the assembly of C3/C5-convertase on the IC,which are subsequently converted into iC3b-iC3b-IgG by the complementregulators. Further processing of iC3b can occur through interactionwith these regulators, to produce C3d and C3g. ICs tagged with variousforms of C3 have been detected in a variety of autoimmune disease, andC3d-IC levels in particular have been shown to correlate directly withdisease activity level in SLE. See Toong C, Adelstein S, Phan T G (2011)Int J Nephrol Renovasc Dis “Clearing the complexity: immune complexesand their treatment in lupus nephritis,” 4:17-28, which is herebyincorporated by reference in its entirety and in particular all figures,legends and disclosure related to models of DNA-anti-DNA immune complexgeneration and glomerular damage in lupus nephritis and potentialtherapeutic targets. See also Sekita K, Doi T, Muso E, Yoshida H,Kanatsu K, Hamashima Y (1984) Clin Exp Immunol “Correlation of C3dfixing circulating immune complexes with disease activity and clinicalparameters in patients with systemic lupus erythematosus,”55(3):487-494, which is hereby incorporated by reference in its entiretyand in particular all figures, legends and disclosure related to CIClevels and anti-C3d assays from patients with various diseases.

The natural receptor for C3d is the complement receptor 2 (CR2), alsoknown as CD21, expressed on the surface of B cells. CR2 serves as a linkto from the innate to the adaptive immune system, and in healthyconditions, the interaction of C3d-tagged immune complexes leads to anamplified B cell/antibody response to the offending antigen.Unfortunately, in autoimmune diseases this amplification can lead tocontinuation of an auto-antibody response to autoantigen, furtherexacerbating the disease.

Soluble CRs and CR-Fc fusions have been described for therapeuticpurposes. These include CR1, CR2-Fc (U.S. Pat. No. 6,458,360), CR2-fH(CR2-factor H), and others. However, while these approaches generallyblock interaction of C3-tagged ICs with their associated receptors, theydo not necessarily remove the immune complexes from circulation. Most ofthe complement receptors and regulatory proteins are composed of one ormore so-called short complement repeat (SCR) domains, also calledcomplement control protein (CCP) modules or Sushi domains. Typically,only a subset of the domains is involved in direct recognition of theassociated complement fragment ligand. For example, it has beendemonstrated that only the first two SCRs of CR2 are essential for C3dbinding. The SCR domains are stable and well-behaved, making themsuitable for use in the development of therapeutic proteins.

Of further relevance to the present invention relates to the mechanismsof hemophilia. One issue with hemophiliacs is the effect that FactorVIII (FVIII (not to be confused with “Fv”)) inhibitors play in disease.Currently, these FVIII inhibitors (generally FVIII antibodies, as shownin FIG. 28) are a huge problem for hemophiliacs.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention provides compositionsand methods for rapidly lowering the serum concentration of an antigenin a patient comprising administering an antibody comprising a variableregion that binds the antigen and a variant Fc domain comprising anamino acid substitution as compared to a parent Fc domain wherein saidvariant Fc domain binds human FcγRIIb with increased affinity ascompared to said parent Fc domain. These antibodies bind to said antigento form an antibody-antigen complex and said complex is cleared at leasttwo fold faster than the antigen alone.

In a further aspect, the present invention provides compositions andmethods for lowering the free antigen in a patient comprisingadministering an antibody comprising a variable region that binds theantigen and a variant Fc domain comprising an amino acid substitution ascompared to a parent Fc domain wherein said variant Fc domain bindsFcγRIIb with increased affinity as compared to said parent Fc domain.The administration results in the concentration of said free antigendecreasing at least 50% more rapidly than the decrease in concentrationseen with an antibody comprising the parent Fc domain.

In a further aspect, the present invention provides compositions andmethods for differentially clearing an antibody-antigen complex in apatient compared to antibody alone, comprising administering an antibodycomprising a variable region that binds the antigen and a variant Fcdomain comprising an amino acid substitution as compared to a parent Fcdomain wherein said variant Fc domain binds FcγRIIb with increasedaffinity as compared to said parent Fc domain. These antibodies bind tothe antigen to form an antibody-antigen complex and the complex iscleared at least two fold faster than the antigen alone.

In one embodiment and in accordance with any of the above, the inventionprovides methods wherein the variant Fc domain comprises amino acidsubstitutions selected from the group consisting of those of FIG. 30,FIG. 47, and FIG. 48.

In a further embodiment and in accordance with any of the above, thepresent invention provides compositions and methods wherein the variantFc domain further comprises amino acid substitutions selected from thegroup consisting of 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.

In a yet further embodiment and in accordance with any of the above,according to any previous claim, the present invention providescompositions and methods wherein the increased affinity seen with thevariant Fc domain is at least a 5-fold or a 10-fold increase as comparedto the parent Fc domain as measured by a Biacore assay.

In a still further embodiment and in accordance with any of the above,the present invention provides compositions and methods that lower serumconcentration of antigen, where the antigen is selected from the groupconsisting of IgE, oxoLDL, and FVIII inhibitor.

In a still further embodiment and in accordance with any of the above,the present invention provides compositions and methods in which theantibody includes a variable region VH domain that comprises a CDR1 ofSEQ ID NO:2, a CDR2 of SEQ ID NO:3 and a CDR3 of SEQ ID NO:4 and avariable region VL domain that comprises a CDR1 of SEQ ID NO:6, a CDR2of SEQ ID NO:7 and a CDR3 of SEQ ID NO:8.

In a still further embodiment and in accordance with any of the above,the present invention provides compositions and methods in which theantibody includes a variable region VH domain that comprises a CDR1 ofSEQ ID NO:18, a CDR2 of SEQ ID NO:19 and a CDR3 of SEQ ID NO:20 and avariable region VL domain that comprises a CDR1 of SEQ ID NO:22, a CDR2of SEQ ID NO:23 and a CDR3 of SEQ ID NO:24.

In a still further embodiment and in accordance with any of the above,the present invention provides compositions and methods in which thevariant Fc domain comprises amino acid substitutions selected from thegroup consisting of S267E, S267D, L328F, P238D, S267E/L328F,G236N/S267E, G236D/S267E.

In a further aspect, the present invention provides a method of rapidlylowering the serum concentration of an antigen in a patient, where themethod includes the step of: administering an Fc fusion proteincomprising: (i) a binding moiety that binds the antigen; and (ii) avariant Fc domain comprising an amino acid substitution as compared to aparent Fc domain, wherein the variant Fc domain binds FcγRIIb withincreased affinity as compared to the parent Fc domain and the Fc fusionprotein binds to the antigen to form a protein-antigen complex that iscleared at least two fold faster than the antigen alone.

In a further aspect, the present invention provides compositions andmethods for lowering the free antigen in a patient comprisingadministering an Fc fusion protein comprising a binding moiety thatbinds the antigen and a variant Fc domain comprising an amino acidsubstitution as compared to a parent Fc domain wherein said variant Fcdomain binds FcγRIIb with increased affinity as compared to said parentFc domain. The administration results in the concentration of said freeantigen decreasing at least 50% more rapidly than the decrease inconcentration seen with an antibody comprising the parent Fc domain.

In a further aspect, the present invention provides compositions andmethods for clearing an antibody-antigen complex in a patient comparedto antibody alone, by administering an Fc fusion protein comprising: (i)a binding moiety that binds to the antigen; and (ii) a variant Fc domaincomprising an amino acid substitution as compared to a parent Fc domain,wherein the variant Fc domain binds FcγRIIb with increased affinity ascompared to said parent Fc domain; and wherein the Fc fusion proteinbinds to said antigen to form a protein-antigen complex and said complexis cleared at least two fold faster than the protein alone.

In a further embodiment in accordance with any of the above, the methodsand compositions of the invention include the use of a fusion proteincontaining a binding moiety that has a sequence selected from FIG. 33Aor 33B.

In a further embodiment in accordance with any of the above, the methodsand compositions of the invention include the use of an Fc fusionprotein that has a first monomer and a second monomer, and the firstmonomer comprises the sequence shown in FIG. 33C and the second monomerhas the sequence shown in FIG. 33D.

In a further embodiment in accordance with any of the above, the methodsand compositions of the invention include the use of an Fc fusionprotein that has a first monomer and a second monomer, and the firstmonomer comprises the sequence shown in FIG. 33E and the second monomerhas the sequence shown in FIG. 33D.

In a yet further embodiment in accordance with any of the above, themethods and compositions of the invention include the use of an Fcfusion protein that has a first domain comprising a CR2 sequence and asecond domain comprising an engineered Fc domain. In still furtherembodiments, the fusion protein sequence is selected from the sequencesdepicted in FIG. 40.

In a further aspect, the present invention provides methods andcompositions for treating an IgE-mediated disease in a patient byrapidly lowering serum concentration of IgE in said patient byadministering an antibody that has (i) a variable region that binds IgE;and (ii) a variant Fc domain comprising an amino acid substitution ascompared to a parent Fc domain, where variant Fc domain binds FcγRIIbwith increased affinity as compared to the parent Fc domain, and wherethe antibody binds to the IgE to form an antibody-IgE complex and thecomplex is cleared at least two fold faster than IgE alone. In certainembodiments, the variant Fc domain comprises amino acid substitutionsselected from the group consisting of S267E, S267D, L238F, P238D,S267E/L328F, G236N/S267E, G236D/S267E. In further embodiments, the IgEmediated disease is selected from the group consisting of: asthma,allergic rhinitis, atopic dermatitis, and food allergy.

In a further aspect, the present invention provides methods andcompositions for treating an autoimmune disorder in a patient by rapidlylowering serum concentration of C3d in the patient by administering arapid clearance molecule comprising: (i) a variable region that bindsC3d; and (ii) a variant Fc domain comprising an amino acid substitutionas compared to a parent Fc domain, where the variant Fc domain bindsFcγRIIb with increased affinity as compared to said parent Fc domain,and where the rapid clearance molecule binds to the C3d to form amolecule-C3d complex and the complex is cleared at least two fold fasterthan C3d alone. In certain embodiments, the variant Fc domain comprisesamino acid substitutions selected from the group consisting of S267E,S267D, L238F, P238D, S267E/L328F, G236N/S267E, G236D/S267E. In furtherembodiments, the autoimmune disorder is selected from the groupconsisting of: systemic lupus erythematosus and rheumatoid arthritis. Inyet further embodiments, the rapid clearance molecule is an antibody oran Fc fusion protein.

In a further aspect, the present invention provides methods andcompositions for treating atherosclerosis in a patient by rapidlylowering serum concentration of oxLDL in the patient by administering arapid clearance molecule that has: (i) a variable region that bindsoxLDL; and (ii) a variant Fc domain comprising an amino acidsubstitution as compared to a parent Fc domain, where the variant Fcdomain binds FcγRIIb with increased affinity as compared to the parentFc domain; and where the rapid clearance molecule binds to the oxLDL toform a molecule-oxLDL complex that is cleared at least two fold fasterthan oxLDL alone. In certain embodiments, the variant Fc domaincomprises amino acid substitutions selected from the group consisting ofS267E, S267D, L238F, P238D, S267E/L328F, G236N/S267E, G236D/S267E. Inyet further embodiments, the rapid clearance molecule is an antibody oran Fc fusion protein.

In a further aspect, the present invention provides methods andcompositions for treating hemophilia in a patient by rapidly loweringserum concentration of FVIII inhibitor in said patient by administeringa rapid clearance molecule comprising (i) a variable region that bindssaid FVIII inhibitor; and (ii) a variant Fc domain comprising an aminoacid substitution as compared to a parent Fc domain, wherein the variantFc domain binds FcγRIIb with increased affinity as compared to theparent Fc domain and wherein the rapid clearance molecule binds to theFVIII inhibitor to form a molecule-inhibitor complex and the complex iscleared at least two fold faster than FVIII inhibitor alone. In certainembodiments, the variant Fc domain comprises amino acid substitutionsselected from the group consisting of S267E, S267D, L238F, P238D,S267E/L328F, G236N/S267E, G236D/S267E. In yet further embodiments, therapid clearance molecule is an antibody or an Fc fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. FIG. 1A Illustrates the novel mechanistic approach forinhibiting IgE+ FcγRIIb+ B cells. Under appropriate stimuli, naive Bcells can differentiate into IgE+ B cells. Engagement of antigen withthe IgE B cell receptor activates these cells, which can thendifferentiate into plasma cells that release circulating IgE. Binding ofcirculating IgE binds to FcεR's, for example on mast cells, basophils,and eosinophils, activates these cells. Release of histamine,prostaglandins, and other chemical mediators ultimately results in theclinical symptoms of allergy and asthma. Omalizumab, having a nativeIgG1 Fc region, is capable of blocking binding of IgE to FcεR. Anti-IgEantibodies with high affinity for FcγRIIb, referred to as Anti-IgE-IIbEin the figure, are capable of not only blocking binding of IgE to FcεR,but also of inhibiting activation of IgE+ B cells by mIgE FcγRIIbcoengagement. FIG. 1B shows the rapid clearance mechanism, outlining thepossible mechanisms of action (MOA): the first is to sequester the freeantigen (in the figure this is IgE), secondly the production of theantigen is suppressed, in the case of IgE, and finally the complex ofthe antigen-antibody is cleared rapidly.

FIG. 2. Biacore surface plasmon resonance sensorgrams showing binding ofFc variant anti-CD19 antibodies to human FcγRIIb.

FIG. 3. Affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore. The graph shows the log(K_(A)) for binding ofvariant and WT IgG1 antibodies to human FcγRI (I), H131 FcγRIIa (H IIa),FcγRIIb (IIb), and V158 FcγRIIIa (Villa). Binding of G236D/S267E andS267E/L328F to V158 FcγRIIIa was not detectable. Binding of G236R/L328R(Fc-KO) to all receptors tested was not detectable.

FIG. 4. Affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore surface plasmon resonance. The table providesequilibrium K_(D)'s for binding of variant and WT IgG1 antibodies tohuman FcγRI, H131 FcγRIIa FcγRIIb, and V158 FcγRIIIa, and the foldbinding for each relative to native (WT) IgG1. n.d.=not detectable.

FIGS. 5A-C. Amino acid sequences of the heavy (VH) and light (VL) chainvariable regions and CDRs of anti-IgE antibodies. CDR boundaries weredefined as described previously based on a structural alignment ofantibody variable regions (Lazar et al., 2007, Mol Immunol44:1986-1998).

FIG. 6. Amino acid sequences of the heavy and light chain WT and variantconstant regions.

FIG. 7. Amino acid sequences of anti-IgE full length antibodies that maybe used to target IgE+ B cells.

FIG. 8. Table of affinity data for binding of WT and variant anti-IgEantibodies to the IgE Fc region and FcγRIIb.

FIG. 9. Plot of affinity data for binding of WT and variant anti-IgEantibodies to the IgE Fc region and FcγRIIb.

FIG. 10. IgE ELISA using commercial (MabTech) and in-house (Omalizumaband MaE11) anti-IgE antibodies as capture reagents.

FIG. 11. The variable region of the anti-IgE antibody omalizumab doesnot compete with MabTech capture antibody for IgE detection in the ELISAprotocol.

FIG. 12. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity, but not antibodies lackingFcγR binding (Fc variant G236R/L328R) or lacking binding to IgE(motavizumab). The plot shows the concentration of IgE released fromPBMCs after 12 days incubation with IL-4, anti-CD40 (α-CD40) agonistantibody, and varying concentrations of the antibodies shown.

FIG. 13. Variant anti-IgE antibodies do not inhibit class-switched IgG2+B cells. The plot shows the concentration of IgG2 released from PBMCsafter 12 days incubation with IL-4, α-CD40, and varying concentrationsof the antibodies shown.

FIG. 14. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity. The plot shows theconcentration of IgE released from PBMCs after 14 days incubation withIL-4, anti-CD40 (α-CD40) agonist antibody, and varying concentrations ofthe antibodies shown. Data were normalized to the lowest concentrationof antibody.

FIG. 15. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity. The plot shows theconcentration of IgE released from PBMCs after 14 days incubation withIL-4, anti-CD40 (α-CD40) agonist antibody, anti-CD79b BCR cross-linkingantibody, and varying concentrations of the antibodies shown. Data werenormalized to the lowest concentration of antibody.

FIG. 16. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity. The plot shows theconcentration of IgE released from PBMCs after 14 days incubation withIL-4, anti-CD40 (α-CD40) agonist antibody, anti-mu BCR cross-linkingantibody, and varying concentrations of the antibodies shown. Data werenormalized to the lowest concentration of antibody.

FIG. 17. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity. The plot shows theconcentration of IgE released from PBMCs after 14 days incubation withIL-4, anti-CD40 (α-CD40) agonist antibody, anti-CD79b BCR cross-linkingantibody, and varying concentrations of the antibodies shown.

FIG. 18. Inhibition of class-switched IgE+ B cells with variant anti-IgEantibodies enhanced for FcγRIIb affinity. The plot shows theconcentration of IgE released from PBMCs after 14 days incubation withIL-4, anti-CD40 (α-CD40) agonist antibody, anti-mu BCR cross-linkingantibody, and varying concentrations of the antibodies shown.

FIG. 19. Protocol for huPBL-SCID in vivo study to test activity ofanti-IgE antibodies. The indicated days reflect the number of days afterengraftment of PBMCs from a donor testing positive for IgE antibodiesspecific for Der p 1. Derp1 vacc. indicates vaccination with Der p 1antigen.

FIG. 20. Total serum IgG levels from the huPBL-SCID in vivo model foreach treatment group. The indicated days (7, 23, and 37) reflect theblood draws outlined in the protocol in FIG. 19. PBS indicates theuntreated vehicle group, Omalizumab indicates the group treated withOmalizumab_IgG1, and the 3 H1 L1 MaE11 groups indicate groups treatedwith humanized MaE11 comprising either a WT IgG1 (IgG1), S267E/L328Fvariant (IIbE), or G236R/L328R (Fc-KO) Fc region.

FIG. 21. Total serum IgE levels from the huPBL-SCID in vivo model foreach treatment group. The indicated days (7, 23, and 37) reflect theblood draws outlined in the protocol in FIG. 19. PBS indicates theuntreated vehicle group, Omalizumab indicates the group treated withOmalizumab_IgG1, and the 3 H1 L1 MaE11 groups indicate groups treatedwith humanized MaE11 comprising either a WT IgG1 (IgG1), S267E/L328Fvariant (IIbE), or G236R/L328R (Fc-KO) Fc region. The limit ofquantitation for the ELISA method was 31.6 ng/mL; samples that werebelow this limit were reported as 31.6 ng/mL in the plot.

FIG. 22 A-C. Data from a chimp study of XmAb7195, described herein, thatshows a rapid and unprecedented reduction in total IgE. The dosage was asingle 5 mg/kg dose, mean baseline IgE level is ˜3 ug/ml. LLOQ is thelower limit of quantification. This contrasts with a known Xolair sideeffect that the concentration of total IgE is increased uponadministration.

FIG. 23. A scatter plot of calculated half-lives for individual micetreated with variant anti-IgE antibodies (IIbE=S267E/L328F).

FIG. 24. Serum total IgE concentration as a function of time in humanFcγRIIb transgenic mice treated with anti-mouse IgE antibodies. Thelower limit of quantification of the IgE assay was 13 ng/ml.

FIG. 25. Plot of test article half-life in human FcγRIIb transgenic miceversus FcγRIIb affinity. A direct relationship is observed.

FIG. 26. Plot in vitro internalization of antibody:IgE complexes intoLSEC isolated from FcγRIIb transgenic mice.

FIG. 27. Liver and heart distribution of ⁸⁹Zr-IgE upon co-administrationof saline, XmAb7195 (S267E/L328F), or XENP6782 (IgG1).

FIG. 28. A Factor VIII fusion embodiment to “scrub” FVIII inhibitorantibodies prior to FVIIIa replacement dosing.

FIG. 29. An illustration of primary structure and domain organization ofFVIII.

FIG. 30A-B. List of suitable Fc domain FcγRIIb amino acid substitutionsfor increased FcγRIIb binding.

FIG. 31. The structure of B-domain deleted human Factor VIII. DomainsA1, A2, A3, C1, and C2 are indicated.

FIG. 32. Diagram showing Factor VIII inhibitor scrubber constructsconsisting of FVIII domains A2 and C2 fused to a rapid clearance IIb Fc.

FIG. 33A-E. Sequences of Factor VIII inhibitor constructs.

FIG. 34. Reducing and non-reducing SDS-PAGE of Factor VIII inhibitorscrubber constructs FVIII_A2_C220S/S267E/L328F andFVIII_C2_C220S/S267E/L328F.

FIG. 35. Size-exclusion chromatography of Factor VIII inhibitor scrubberconstructs FVIII_A2_C220S/S267E/L328F and FVIII_C2_C220S/S267E/L328F.

FIG. 36A-D. Affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore surface plasmon resonance. FIG. 36A is a tablelisting the dissociation constant (Kd) for binding anti-CD19 variantantibodies to human FcγRI, FcγRIIa (131R), FcγRIIa (131H), FcγRIIb,FcγRIIa (158V), and FcγRIIIa (158F). FIG. 36B is a continuation of thelist in FIG. 36A. FIG. 36C is a continuation of the list in FIG. 36A andFIG. 36B. FIG. 36D is a continuation of the list in FIG. 36A, FIG. 36B,and FIG. 36C. Multiple observations have been averaged. n.d.=nodetectable binding.

FIG. 37A-D. Fold affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore surface plasmon resonance. FIG. 37A is a tablelisting the fold improvement or reduction in affinity relative to WTIgG1 for binding of anti-CD19 variant antibodies to human FcγRI, FcγRIIa(131R), FcγRIIa (131H), FcγRIIb, FcγRIIIa (158V), and FcγRIIa (158F).FIG. 37B is a continuation of the list in FIG. 37A. FIG. 37C is acontinuation of the list in FIG. 37A and FIG. 37B. FIG. 37D is acontinuation of the list in FIG. 37A, FIG. 37B, and FIG. 37C.Fold=KD(Native IgG1)/KD(variant). n.d.=no detectable binding.

FIG. 38. General overview of the CR2-IIbE embodiment, the “immunecomplex scrubber” embodiment. As shown, the “rapid clearance” mechanism,utilizing a CR2-Fc fusion, wherein the Fc component of the fusionprotein has increased FcγRIIb binding as compared to a wild-type Fcdomain (particularly an Fc region from a human IgG1, IgG2, IgG3 or IgG4)and the CR component is as described herein.

FIGS. 39A-C. Binding data of CR2-Fc constructs.

FIGS. 40A-F. Sequences for the CR embodiments of the invention.

FIG. 41. Schematic describing the generation of atherosclerosis viamacrophage uptake of oxLDL and its prevention by Fc-containingoxLDL-binding proteins with enhanced FcγRIIb affinity.

FIG. 42. Amino acid sequences for oxLDL-binding proteins.

FIG. 43A-B. Amino acid sequences for Fc-containing oxLDL-bindingproteins.

FIG. 44. Size-exclusion chromatograms for expressed and purifiedFc-containing oxLDL-binding proteins.

FIG. 45. Amino acid sequences for Fc-containing oxLDL-binding proteinswith enhanced FcγRIIb affinity.

FIG. 46. Amino acid sequences for humanized variable regions derivedfrom the EO6 parental antibody.

FIG. 47A-D. List of a variety of suitable Fc domain FcγRIIb amino acidsubstitutions for increased FcγRIIb binding.

FIG. 48A-B. Matrix of possible combinations of FcγRIIb variants, FcRnvariants, Scaffolds, Fvs and combinations, with each variant beingindependently and optionally combined from the appropriate sourceLegend: Legend A are suitable FcRn variants: 434A, 434S, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E, 259I/308F/428L. Legend B are suitable scaffolds andinclude IgG1, IgG2, IgG3, IgG4, and IgG1/2. Sequences for such scaffoldscan be found for example in US Patent Publication No. 2012/0128663,published on May 24, 2012, which is hereby incorporated by reference inits entirety for all purposes and in particular for all teachings,figures and legends related to scaffolds and their sequences. Legend Care suitable exemplary target antigens: IgE, IL-4, IL-6, IL-13, TNFα,MCP-1, RANTES, TARC, MDC, VEGF, HGF, and NGF, immune complexes, FVIIIinhibitors, LDL, oxidized LDL (OxLDL), Lp(a), SOST, and DKK1. Legend Dreflects the following possible combinations, again, with each variantbeing independently and optionally combined from the appropriate sourceLegend: 1) FcγRIIb variants plus FcRn variants; 2) FcγRIIb variants plusFcRn variants plus Scaffold; 3) FcγRIIb variants plus FcRn variants plusScaffold plus Fv; 4) FcγRIIb variants plus Scaffold 5) FcγRIIb variantsplus Fv; 6) FcRn variants plus Scaffold; 7) FcRn variants plus Fv; 8)Scaffold plus Fv; 9) FcγRIIb variants plus Scaffold plus Fv; and 10)FcγRIIb variants plus FcRn variants plus Fv.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Overview of Invention

The in vivo pharmacokinetic properties of therapeutic antibodies can bealtered through modification of their Fc domain. Such modifications mayinclude amino acid substitutions, deletions, or additions as well asother modifications such as chemical modifications. In the presentinvention, modifications that increase affinity of molecules such asantibodies for the inhibitory Fc receptor FcγRIIb (CD32b) are utilizedto facilitate rapid in vivo clearance of complexes comprising theantigen and the molecule of the invention. Incorporation of theIIb-enhancing affinity modifications (also referred to herein as“FcγRIIb variants” or “FcγRIIb variations” or grammatical equivalentsthereof) into various antibodies leads to a novel phenomenon whereby theantibody-target complex is cleared extremely rapidly while the antibodyalone retains a reasonably long half-life. Although much of thediscussion herein is directed to antibodies for the sake of clarity, itwill be appreciated that discussion of the FcγRIIb variants describedherein are applicable to any of the rapid clearance molecules describedherein, including polypeptides, antibodies, and Fc fusion proteins.

The present invention provides methods of rapidly lowering the serumconcentration of an antigen in a subject by administering an antibodythat has both a variable region that binds the antigen and a variant Fcdomain that binds the FcγRIIb receptor with increased affinity ascompared to an un-modified Fc domain. Without being bound by theory, itappears that an antibody of the invention binds to the antigen to forman antibody-antigen complex that is cleared more rapidly than theunbound antigen. Thus, the free antigen concentration in the patient,e.g. the serum concentration of free antigen in the patient, is rapidlydecreased. In other words, the antigen-antibody complex isdifferentially cleared (e.g. clearance of complex/clearance of antigenratio is greater than 1). In some cases, the methods and compositions ofthe present invention clear an antibody-antigen complex at least 2, 5,10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300-foldfaster than clearance of the antigen alone. In certain cases the methodsand compositions of the present invention clear an antibody-antigencomplex 5-500, 50-450, 100-400, 200-350, 100-200-fold faster thanclearance of the antigen alone. In other cases, clearance rates of 25×faster than antigen alone, 50×, 75× and 100× or more are provided bymethods and compositions of the present invention. In some cases, themethods and compositions of the present invention decrease the clearancerate of an antibody-antigen complex by at least 30%, 40%, 50%, 60%, 70%,80% 90%, 95%, or 99% as compared to the clearance rate of the antigenalone. In some cases, the methods and compositions of the presentinvention decrease the clearance rate of an antibody-antigen complex byat least 30%, 40%, 50%, 60%, 70%, 80% 90%, 95%, or 99% as compared tothe clearance rate mediated by an antibody comprising a parent(un-modified) Fc domain.

Thus, compositions of the present invention include such “rapidclearance” molecules (also referred to as “scrubbers”) of the presentinvention that lead to clearance of the antibody-antigen complex morerapidly than the unbound antigen or antibody alone. Such rapid clearancecompositions are generally polypeptides that comprise two domains: anantigen or ligand binding portion and an Fc domain that exhibitsincreased FcγRIIb binding as compared to a non-engineered Fc region. Insome embodiments, as is further described herein, the rapid clearancemolecules are antibodies, comprising a standard antigen binding Fvregion, and a variant FcγRIIb binding region, e.g. an engineered Fcregion. In alternative embodiments, the rapid clearance molecule is anFc fusion protein, with a binding ligand or receptor as one domain (e.g.a CR domain) and an Fc region with increased FcγRIIb binding. Inaddition to the faster clearance rates described above as compared toclearance of antigen alone, rapid clearance molecules of the inventionfurther clear the antigen-containing complex more rapidly than IgGantibodies with the same selectivity would mediate.

Application of different FcγRIIb-enhancing Fc amino acid substitutionswith varying affinities to the FcγRIIb receptor (e.g. S267E, S267D,L328F, P238D, S267E/L328F, G236N/S267E, and G236D/S267E, as furtherdescribed herein) can allow some “tuning” of how fast the complexantigen is cleared while maintaining significant half life of the rapidclearance composition of the invention (including antibodies). That is,different amino acid substitutions that alter FcγRIIb binding affinitymay lead to different balances between the complex clearance rate andthe antibody clearance rate, allowing for tailoring toward optimaltherapeutic profile and dosing. This tuning may be accomplished by usingamino acid substitutions in the Fc domain that increase binding toFcγRIIb as compared to the parent Fc domain. This increase in bindingmay be tuned by using Fc variants with 1-100, 5-90, 10-80, 15-70, 20-60,30-50, 10-20 fold greater affinity as compared to the parent Fc domain.This increase in binding may also be tuned by using Fc variants with50-200, 60-190, 70-180, 80-170, 90-160, 100-150, 110-140, 120-130,50-100 greater affinity as compared to the parent Fc domain. In somecases, affinity is measured by Biacore as described in Example 2.

In certain cases, molecules of the invention incorporate FcγRIIbreceptor variants that can range from very tight differential binding toFcγRIIb to variants that display increased (as compared to wild type Fcdomains) binding affinity but at a lower level. For example, very tight(or heavy) binding to FcγRIIb receptor may include FcγRIIb variants thatshow at least 50, 75, 100, 125, 150, 175, 200, 225, 250-fold greateraffinity to FcγRIIb receptor as compared to the parent Fc domain. Incontrast, a lower level (or light, also referred to herein as “lite”)increase in binding may include FcγRIIb variants that show no more than50, 40, 30, 20, 10, 5-fold greater affinity to FcγRIIb receptor ascompared to the parent Fc domain.

The effects of molecules of the invention may be further tuned bycombining amino acid substitutions that alter FcγRIIb binding affinitywith amino acid substitutions that affect binding to FcRn. Proteins withamino acid substitutions that affect binding to FcRn (also referred toherein as “FcRn variants”) may in certain situations also increase serumhalf-life in vivo as compared to the parent protein. As will beappreciated, any combination of Fc and FcRn variants may be used to tuneclearance of the antigen-antibody complex. Suitable FcRn variants thatmay be combined with any of the Fc variants described herein includewithout limitation 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F,436I/428L, 436I/434S, 436V/434S, 436V/428L, 252Y, 252Y/254T/256E, and259I/308F/428L.

Without being bound by theory, it appears that the accelerated clearanceof antibodies containing amino acid substitutions that confer highaffinity (as compared to the parent Fc domain) to the inhibitoryreceptor FcγRIIb is likely mediated by interaction withFcγRIIb-expressing cells, possibly liver sinusoidal endothelial cells.In addition, it appears that the accelerated clearance is due to theclearance of the antigen-antibody complex via interactions with theFcγRIIb receptor.

In addition, unexpectedly, administration of antibodies including theFcγRIIb binding affinity variants described herein leads to nearinstantaneous drops in total antigen levels, whereas administration ofother antibodies to the antigen that lack modifications that lead toincreased FcγRIIb binding affinity often lead to increases in totalantigen levels. Furthermore, the greater reduction in total antigenlevels seen with antibodies with increased FcγRIIb binding affinityleads to superior reduction of free antigen relative to levels seen withantibodies that lack the FcγRIIb variants.

In addition, in some cases, compositions and methods of the inventionprovide sufficient increased affinity to the FcγRIIb receptor to allowfor rapid clearance of the antibody-antigen complex while allowingappropriate serum half lives of the unbound antibodies.

The invention is exemplified in the case of XmAb7195. XmAb7195 is ananti-IgE antibody that sequesters IgE and prevents its interaction withFceR1 on mast cells and basophils. The variable region is similar to thevariable region of omalizumab (Xolair, an anti-IgE antibody). TheXmAb7195 Fc domain was engineered with the S267E/L328F substitutions(Kabat numbering) to confer high affinity to human FcγRIIb. A single 5mg/kg dose of XmAb7195 was administered via intravenous injection tochimpanzees (n=3) and its effects on free and total IgE were compared tothat of omalizumab (anti-IgE with native IgG1 Fc domain). As shown inFIG. 22, the XmAb7195 antibody had a relatively short half-life ofapproximately 2 days, and the omalizumab exhibited a longer half-life ofapproximately 11 days.

Unexpectedly, the XmAb7195 antibody also led to a near-instantaneousdrop of total IgE levels, whereas the omalizumab treatment led to anincrease in total IgE (as observed in humans treated with omalizumab).The XmAb7195-IgE complexes exhibited greatly accelerated clearancepresumably via their interaction with FcRIIb. Furthermore, the greaterreduction of total IgE using XmAb7195 led to superior reductions of freeIgE relative to omalizumab-treated animals. PK/PD simulations suggestthat the half-life of the XmAb7195/IgE complexes are on the order of 1hour—versus an 8 day half-life reported for omalizumab/IgE complexes.Furthermore, the present invention also suggests that rapid recovery ofthe antigen can occur after cessation of antibody administration.

This surprising and unexpected result, e.g. that adding FcγRIIb variantsto existing antibodies can rapidly clear antigen in patients leads to anumber of useful applications. Any therapeutic target antigen system inwhich rapid clearance of the antigen is desired can be subjected to thepresent invention. For example, disease systems in which the antigenload is high find particular use in the present invention. Similarly,disease systems where rapid recovery of the antigen is desired afterantibody administration can be treated with the antibodies of theinvention. For example, during the use of TNF antibody inhibitors,patients frequently get infections. Withdrawing the antibody treatmentwill allow rapid recovery of the TNF to fight the infection. Similarly,these antibodies can be used to treat pathogen infection when rapidpathogen clearance is desired (for example, when a patient scheduled forsurgery gets an infection, the present invention can be used to clearthe infection rapidly, the therapeutic antibody rapidly clears as welland surgery can progress). In addition, these antibodies may beparticularly useful in situations where existing antibodies do notneutralize the antigen, or where pathogens evolve to evadeneutralization.

In addition, the invention finds use in the treatment of hemophiliacs.One issue with hemophiliacs is the effect that Factor VIII (FVIII (notto be confused with “Fv”)) inhibitors play in disease. Currently, theseFVIII inhibitors (generally FVIII antibodies, as shown in FIG. 28) are ahuge problem for hemophiliacs. The present invention works as generallyoutlined in FIGS. 28 and 29. Fc fusion proteins, comprising an Fc domainwith FcγRIIb amino acid variants, fused to FVIIIa components as outlinedherein, will sequester the inhibitor antibodies, rapidly clear theinhibitor antibodies, and will inhibit FVII-reactive B cells (toprohibit the further production of the inhibitors).

The rapid clearance mechanisms of the present invention are also used toremove oxidized low-density lipoprotein (oxidized LDL or oxLDL) from theblood. OxLDL is a key facilitator of atherosclerosis via macrophageuptake and foam cell formation (see FIG. 42). In this embodiment,increased affinity for the inhibitory Fc receptor FcγRIIb (CD32b) isutilized to facilitate rapid in vivo clearance of oxLDL via theirinteraction with Fc-containing oxLDL-binding proteins. Incorporation ofthe IIb-enhancing affinity substitutions into various Fc-containingoxLDL-binding proteins leads to a novel phenomenon whereby the complexis cleared extremely rapidly. Application of different IIb-enhancingsubstitutions (including without limitation S267E, S267D, L328F, P238D,S267E/L328F, G236N/S267E, and G236D/S267E, which are useful for all therapid clearance molecules herein) may lead to different balances betweenthe complex clearance rate and the Fc-containing oxLDL-binding proteinclearance rate, allowing for tailoring toward optimal therapeuticprofile and dosing.

In addition, the invention finds use in a variety of diseases orsituations where plasmapheresis is typically applied to clear the bodyof pathogens, autoantibodies, or other pathogenic factors. Such diseasesinclude, but are not limited to, the following: Guillain-Barré syndrome;Chronic inflammatory demyelinating polyneuropathy; Goodpasture'ssyndrome; Hyperviscosity syndromes: Cryoglobulinemia; Paraproteinemia;Waldenström macroglobulinemia; Myasthenia gravis; Thromboticthrombocytopenic purpura (TTP)/hemolytic uremic syndrome; Wegener'sgranulomatosis; Lambert-Eaton Syndrome; Antiphospholipid AntibodySyndrome (APS or APLS); Microscopic polyangiitis; Recurrent focal andsegmental glomerulosclerosis in the transplanted kidney; HELLP syndrome;PANDAS syndrome; Refsum disease; Behcet syndrome; HIV-relatedneuropathy; Graves' disease in infants and neonates; Pemphigus vulgaris;Multiple sclerosis; Rhabdomyolysis; Toxic Epidermal Necrolysis (TEN).That is, by using FcγRIIb antibodies with variable regions specific tothese antigens, clearance of the antigens in a rapid manner can occur.

DEFINITIONS

Described herein are several definitions. Such definitions are meant toencompass grammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding is the double variant 236R/328R, and236R and 328R separately as well.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. The role of high affinitybinding to FcγRIIIa to ADCC activity is well established. In some cases,as described herein, amino acid substitutions in the Fc domain can beused to increase or decrease binding to one or more of the FcγRreceptors, as is generally outlined in US Publication 2006/0024298,hereby incorporated by reference in its entirety and in particular forthe amino acid substitutions disclosed therein, FIG. 41 as well as theother figures and their accompanying legends in particular. In addition,for some embodiments outlined herein, it may be desirable to ablatebinding to one or more of the FcγR receptors. For example, the L328Fvariant ablates FcγRIIIa binding, such that ADCC mechanisms are nottriggered. In addition, significant ablatement of FcγR binding to allreceptors can be accomplished using 236R/328R variants.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith a different amino acid. In particular, in some embodiments, thesubstitution is to an amino acid that is not naturally occurring at theparticular position, either not naturally occurring within the organismor in any organism. For example, the substitution E272Y refers to avariant polypeptide, in this case an Fc variant, in which the glutamicacid at position 272 is replaced with tyrosine. For clarity, a proteinwhich has been engineered to change the nucleic acid coding sequence butnot change the starting amino acid (for example exchanging CGG (encodingarginine) to CGA (still encoding arginine) to increase host organismexpression levels) is not an “amino acid substitution”; that is, despitethe recombinant creation of a new gene encoding the same protein, if theprotein has the same amino acid at the particular position that itstarted with, it is not an amino acid substitution. By “amino acidinsertion” or “insertion” as used herein is meant the addition of anamino acid at a particular position in a parent polypeptide sequence. By“amino acid deletion” or “deletion” as used herein is meant the removalof an amino acid at a particular position in a parent polypeptidesequence.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (υ), delta (δ), gamma (γ), sigma (σ), and alpha(α) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE,and IgA (IgA1 and IgA2) isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position.

By “CD32b+ cell” or “FcγRIIb+ cell” as used herein is meant any cell orcell type that expresses CD32b (FcγRIIb). CD32b+ cells include but arenot limited to B cells, plasma cells, dendritic cells, macrophages,neutrophils, mast cells, basophils, or eosinophils.

By “IgE+ cell” as used herein is meant any cell or cell type thatexpresses IgE. In preferred embodiments of the invention, IgE+ cellsexpress membrane-anchored IgE (m IgE). IgE+ cells include but are notlimited to B cells and plasma cells.

By “CDC” or “complement dependent cytotoxicity” as used herein is meantthe reaction wherein one or more complement protein components recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell.

By “molecule” or grammatical equivalents is meant a bifunctionalmolecule capable of binding both antigen and FcγRIIb wherein the Kd forbinding of the molecule to FcγRIIb is less than about 100 nM on a cellsurface resulting in simultaneous binding of both the antigen to whichthe antibody is directed and FcγRIIb.

By “constant region” of an antibody as defined herein is meant theregion of the antibody that is encoded by one of the light or heavychain immunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Cκ) or lambda (Cλ) light chains. Theconstant light chain typically comprises a single domain, and as definedherein refers to positions 108-214 of Cκ or Cλ, wherein numbering isaccording to the EU index. By “constant heavy chain” or “heavy chainconstant region” as used herein is meant the region of an antibodyencoded by the mu, delta, gamma, alpha, or epsilon genes to define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For fulllength IgG antibodies, the constant heavy chain, as defined herein,refers to the N-terminus of the CH1 domain to the C-terminus of the CH3domain, thus comprising positions 118-447, wherein numbering isaccording to the EU index.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC and ADCP, and complement-mediated effectorfunctions such as CDC.

By “effector cell” as used herein is meant a cell of the immune systemthat expresses one or more Fc and/or complement receptors and mediatesone or more effector functions. Effector cells include but are notlimited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γδ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the VH, CH1, VH, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody or antibody fragment.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain and in some cases, part of thehinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3)and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, as described below.

By “Fc polypeptide” as used herein is meant a polypeptide that comprisesall or part of an Fc region. Fc polypeptides include antibodies, Fcfusions, isolated Fcs, and Fc fragments. Immunoglobulins may be Fcpolypeptides.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to Fc. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) as used in the prior art(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200, both hereby entirely incorporated byreference). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general may be any protein, polypeptideor small molecule. The role of the non-Fc part of an Fc fusion, i.e.,the fusion partner, is to mediate target binding, and thus it isfunctionally analogous to the variable regions of an antibody. Virtuallyany protein or small molecule may be linked to Fc to generate an Fcfusion. Protein fusion partners may include, but are not limited to, thetarget-binding region of a receptor, an adhesion molecule, a ligand, anenzyme, a cytokine, a chemokine, or some other protein or proteindomain. Small molecule fusion partners may include any therapeutic agentthat directs the Fc fusion to a therapeutic target. Such targets may beany molecule, e.g., an extracellular receptor that is implicated indisease.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,incorporated entirely by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32),FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

By “Fc ligand” or “Fc receptor” as used herein is meant a molecule,e.g., a polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136). Fc ligands may includeundiscovered molecules that bind Fc.

By “full length antibody” as used herein is meant the structure thatconstitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains VL and CL, and each heavy chaincomprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

By “immunoglobulin” herein is meant a protein comprising one or morepolypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies (includingbispecific antibodies) and Fc fusions. Immunoglobulins may have a numberof structural forms, including but not limited to full lengthantibodies, antibody fragments, and individual immunoglobulin domains.

By “immunoglobulin (Ig) domain” as used herein is meant a region of animmunoglobulin that exists as a distinct structural entity asascertained by one skilled in the art of protein structure. Ig domainstypically have a characteristic β-sandwich folding topology. The knownIg domains in the IgG isotype of antibodies are VH Cγ1, Cγ2, Cγ3, VL,and CL.

By “IgG” or “IgG immunoglobulin” or “immunoglobulin G” as used herein ismeant a polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin gamma gene. Inhumans this class comprises the subclasses or isotypes IgG1, IgG2, IgG3,and IgG4.

By “IgE” or “IgE immunoglobulin” or “immunoglobulin E” as used herein ismeant a polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin epsilon gene. IgEmay be membrane-anchored (mIgE), or non-membrane-anchored, also referredto herein as circulating IgE.

By “inhibition” of cells or grammatical equivalents is meant preventingor reducing the activation, proliferation, maturation or differentiationof targeted cells.

By “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

By “modification” herein is meant an alteration in the physical,chemical, or sequence properties of a protein, polypeptide, antibody, orimmunoglobulin. Modifications described herein include amino acidmodifications and glycoform modifications.

By “glycoform modification” or “modified glycoform” or “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Modified glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an Fc variant maycomprise a modified glycoform. Alternatively, modified glycoform mayrefer to the Fc variant that comprises the different carbohydrate oroligosaccharide.

By “parent polypeptide”, “parent protein”, “parent immunoglobulin”,“parent Fc domain”, “precursor polypeptide”, “precursor protein”, or“precursor immunoglobulin” as used herein is meant an unmodifiedpolypeptide, protein, Fc domain, or immunoglobulin that is subsequentlymodified to generate a variant, e.g., any polypeptide, protein orimmunoglobulin which serves as a template and/or basis for at least oneamino acid modification described herein. The parent polypeptide may bea naturally occurring polypeptide, or a variant or engineered version ofa naturally occurring polypeptide. Parent polypeptide may refer to thepolypeptide itself, compositions that comprise the parent polypeptide,or the amino acid sequence that encodes it. Accordingly, by “parent Fcpolypeptide” as used herein is meant an Fc polypeptide that is modifiedto generate a variant Fc polypeptide, and by “parent antibody” as usedherein is meant an antibody that is modified to generate a variantantibody (e.g., a parent antibody may include, but is not limited to, aprotein comprising the constant region of a naturally occurring Ig).

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as described in Kabat. Forexample, position 297 is a position in the human antibody IgG1.

By “polypeptide” or “protein” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “rapid clearance” or grammatical equivalents herein is meant that theantigen-antibody complex composition is cleared from the blood morequickly than either the antigen alone or the antibody, or a complexbetween the antigen and a parent analog of the antibody. As isunderstood in the art, antibodies with different Fvs may have differenthalf lives in serum, so the comparison is to the starting antibody (e.g.an anti-IgE antibody without the IIb variants outlined herein) to theIIb engineered antibody. In general, “rapid clearance” are clearancerates of 25× faster than parent antibody, 50×, 75× and 100× or more. Forexample, as outlined herein, the anti-IgE IIb antibody of the examplesshows a one hour clearance rate in chimps as compared to 2 days ofeither the parent Xolair antibody or an Fc IIb polypeptide that does notcontain a binding moiety for IgE. Similarly, “clearance” can be measuredas a reduction in free target antigen of 10%, 25%, 50% with 90 to 99%percentage of starting serum antigen concentration being a preferredclearance.

By “target antigen” as used herein is meant the molecule that is boundby the variable region of a given antibody, or the fusion partner of anFc fusion. A target antigen may be a protein, carbohydrate, lipid, orother chemical compound. An antibody or Fc fusion is said to be“specific” for a given target antigen based on having affinity for thetarget antigen. A variety of target antigens are listed below.

Virtually any antigen may be targeted by the polypeptides of theinvention, including but not limited to proteins, subunits, domains,motifs, and/or epitopes belonging to the following list of targetantigens, which includes both soluble factors such as cytokines andmembrane-bound factors. Proteins that may be target antigens of theinvention include without limitation: IgE (soluble and/ormembrane-bound), cytokines, e.g., IL-4, IL-6, IL-13, and TNFα;chemokines, e.g., MCP-1, RANTES, TARC, and MDC; growth factors, e.g.,VEGF, HGF, and NGF; also, immune complexes, blood factor inhibitors,e.g. FVIII inhibitors, LDL, oxidized LDL, SOST, and DKK1. Targetantigens may also include without limitation: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin 0, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11 b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor 10a, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein 10b/IIIa (GP 10b/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpi, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Mud), MUC18, Muellerian-inhibitin substance, Mug, MuSK,NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand,TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WI F-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNTSA, WNTSB, WNT6, WNT7A, WNT7B, WNTSA, WNTSB, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

As discussed below, target antigens that find use in the particular useof rapid clearance of antibody-antigen complexes are listed below, andinclude cancer antigens, autoantigens, pathogen antigens, allergyantigens, etc.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide”, “polypeptide variant”, or “variant” as usedherein is meant a polypeptide sequence that differs from that of aparent polypeptide sequence by virtue of at least one amino acidmodification. The parent polypeptide may be a naturally occurring orwild-type (WT) polypeptide, or may be a modified version of a WTpolypeptide. Variant polypeptide may refer to the polypeptide itself, acomposition comprising the polypeptide, or the amino sequence thatencodes it. In some embodiments, variant polypeptides disclosed herein(e.g., variant immunoglobulins) may have at least one amino acidmodification compared to the parent polypeptide, e.g. from about one toabout ten amino acid modifications, from about one to about five aminoacid modifications, etc. compared to the parent. The variant polypeptidesequence herein may possess at least about 80% homology with a parentpolypeptide sequence, e.g., at least about 90% homology, 95% homology,etc. Accordingly, by “Fc variant” or “variant Fc” as used herein ismeant an Fc sequence that differs from that of a parent Fc sequence byvirtue of at least one amino acid modification. An Fc variant may onlyencompass an Fc region, or may exist in the context of an antibody, Fcfusion, isolated Fc, Fc fragment, or other polypeptide that issubstantially encoded by Fc. Fc variant may refer to the Fc polypeptideitself, compositions comprising the Fc variant polypeptide, or the aminoacid sequence that encodes it. By “Fc polypeptide variant” or “variantFc polypeptide” as used herein is meant an Fc polypeptide that differsfrom a parent Fc polypeptide by virtue of at least one amino acidmodification. By “protein variant” or “variant protein” as used hereinis meant a protein that differs from a parent protein by virtue of atleast one amino acid modification. By “antibody variant” or “variantantibody” as used herein is meant an antibody that differs from a parentantibody by virtue of at least one amino acid modification. By “IgGvariant” or “variant IgG” as used herein is meant an antibody thatdiffers from a parent IgG by virtue of at least one amino acidmodification. By “immunoglobulin variant” or “variant immunoglobulin” asused herein is meant an immunoglobulin sequence that differs from thatof a parent immunoglobulin sequence by virtue of at least one amino acidmodification.

By “wild type” or “WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc. has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

Rapid Clearance Molecules

The present invention is directed to the use of rapid clearancemolecules (also referred to herein as “IIb variants”) with high affinityto the FcγRIIb receptor that result in the rapid clearance from serum ofthe antibody-antigen complex, while retaining significant if not all theserum half-life of the unbound antigen or unbound rapid clearancemolecules. In certain embodiments, the rapid clearance molecules of theinvention include antibodies or Fc fusion proteins. Although much of thediscussion herein is in terms of antibodies for ease of discussion, itwill be appreciated that this discussion applies equally to any of therapid clearance molecules described herein.

In general, rapid clearance molecules of the invention generallycomprise a variable region that binds to an antigen and a variant Fcdomain comprising one or more amino acid substitutions as compared to aparent Fc domain such that the variant Fc domain binds FcγRIIb withincreased affinity as compared to the parent Fc domain.

In certain aspects, the rapid clearance (“RC”) antibodies incorporateFcγRIIb receptor variants that can range from very tight differentialbinding to FcγRIIb to variants that display increased (as compared towild type Fc domains) binding affinity but at a lower level. Forexample, very tight (or heavy) binding to FcγRIIb receptor may includeFcγRIIb variants that show at least 50, 75, 100, 125, 150, 175, 200,225, 250-fold greater affinity to FcγRIIb receptor as compared to theparent Fc domain. In contrast, a lower level (or light, also referred toherein as “lite”) increase in binding may include FcγRIIb variants thatshow no more than 50, 40, 30, 20, 10, 5-fold greater affinity to FcγRIIbreceptor as compared to the parent Fc domain. In further embodiments,tighter/heavier binding FcγRIIb variants show 50-300, 60-275, 70-250,80-225, 90-200, 100-175, 110-150-fold greater affinity to FcγRIIbreceptor as compared to the parent Fc domain, whereas lower/lighterbinding FcγRIIb variants show 2-40, 4-35, 6-30, 8-25, 9-20, 10-15-foldgreater affinity to FcγRIIb receptor as compared to the parent Fcdomain. In certain embodiments, affinity is measured using Biacore, forexample as described in Example 2. As discussed herein, the functionalproperties of rapid clearance molecules, including rapid clearanceantibodies, can be tuned using modifications (such as amino acidsubstitutions, deletions or additions) that increase binding to FcγRIIbreceptor by a tighter/heavier or a lower/lighter degree.

As shown herein, the binding affinity of the IIb variants can bemanipulated to result in different clearance/half life ratios. That is,the IIb variant S267E/L328F shows very high affinity binding to FcγRIIbof the variants discussed herein, and also has a faster clearance ratefor antigen-antibody complexes among the variants discussed herein. Thebinding affinity, antibody half lives and clearance rates can beadjusted using high affinity binding variants or lower affinity bindingvariants, e.g. S267E, G236N/S267E, etc.). For example, the bindingaffinity of S267E is about 10× lower than the S267E/L328F variant, witha corresponding increase in half-life of roughly 2×-4× higher. Incertain aspects, higher/heavier binding results in decreases inhalf-life. Thus, clearance rate and half-life can be adjusted byutilizing FcγRIIb-enhancing Fc amino acid substitutions that possessintermediate or lower increases in binding affinity (e.g., thesesubstitutions still result in variants with higher affinity for FcγRIIbreceptor than the parent molecules, but the affinities for thesevariants is not as increased, i.e., is lower/lighter than the heavybinding variants). Correlations between half-life and binding affinitycan be measured as known in the art and discussed herein—see for exampleFIG. 25 and Example 6.

Application of different FcγRIIb-enhancing Fc amino acid substitutionswith varying affinities to the FcγRIIb receptor (e.g. S267E/L328F,G236D/S267E, G236N/S267E, and S267E alone, as further described herein)can allow some “tuning” of how fast the complex antigen is cleared whilemaintaining significant half life of the rapid clearance composition ofthe invention (including antibodies). That is, different amino acidsubstitutions that alter FcγRIIb binding affinity may lead to differentbalances between the complex clearance rate and the antibody clearancerate, allowing for tailoring toward optimal therapeutic profile anddosing. This tuning may be accomplished by using amino acidsubstitutions in the Fc domain that increase binding to FcγRIIb ascompared to the parent Fc domain. This increase in binding may be tunedby using Fc variants with 1-100, 5-90, 10-80, 15-70, 20-60, 30-50,10-20, 5-15, fold greater affinity as compared to the parent Fc domain.This increase in binding may also be tuned by using Fc variants with20-500, 30-400, 40-300, 50-200, 60-190, 70-180, 80-170, 90-160, 100-150,110-140, 120-130, 50-100, 25-75 fold greater affinity to FcγRIIbreceptor as compared to the parent Fc domain.

The effects of molecules of the invention may be further tuned bycombining amino acid substitutions that alter FcγRIIb binding affinitywith amino acid substitutions that affect binding to FcRn. Proteins withamino acid substitutions that affect binding to FcRn (also referred toherein as “FcRn variants”) may in certain situations also increase serumhalf-life in vivo as compared to the parent protein. As will beappreciated, any combination of Fc and FcRn variants may be used to tuneclearance of the antigen-antibody complex. Suitable FcRn variants thatmay be combined with any of the Fc variants described herein that alterbinding to FcγRIIb include without limitation 434A, 434S, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I/434S, 436V/434S, 436V/428L,252Y, 252Y/254T/256E, and 259I/308F/428L.

In further embodiments, combinations of variants that alter binding tothe FcγRIIb are combined with a variety of scaffolds, target antigensand/or FcRn variants to further tune clearance properties or otherfunctional properties (such as binding to FcγRIIa) of the antibodies.Exemplary (non-limiting) combinations are provided in FIG. 48, whichprovides a matrix of possible combinations, with each variants beingindependently and optionally combined from the appropriate sourceLegend: Legend A are suitable FcRn variants: 434A, 434S, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E, 259I/308F/428L. Legend B are suitable scaffolds andinclude IgG1, IgG2, IgG3, IgG4, and IgG1/2. Legend C are suitableexemplary target antigens: IgE (soluble or membrane-bound), IL-4, IL-6,IL-13, TNFα, MCP-1, RANTES, TARC, MDC, VEGF, HGF, and NGF, immunecomplexes, FVIII inhibitors, LDL, oxidized LDL (OxLDL), Lp(a), SOST, andDKK1. Legend D reflects the following possible combinations, again, witheach variant being independently and optionally combined from theappropriate source Legend: 1) FcγRIIb variants plus FcRn variants; 2)FcγRIIb variants plus FcRn variants plus Scaffold; 3) FcγRIIb variantsplus FcRn variants plus Scaffold plus Fv; 4) FcγRIIb variants plusScaffold 5) FcγRIIb variants plus Fv; 6) FcRn variants plus Scaffold; 7)FcRn variants plus Fv; 8) Scaffold plus Fv; 9) FcγRIIb variants plusScaffold plus Fv; and 10) FcγRIIb variants plus FcRn variants plus Fv.Note that any of these combinations may also include any of the FcγRIIbvariants described herein, including those listed in FIGS. 30, 36, 48 aswell as those listed in the first column of FIG. 48. Any of thesecombinations may also include any Fc variants known in the art,including for example variants described in WO 2012/115241;WO2013/125667; U.S. Pat. No. 6,737,056; U.S. Pat. No. 8,435,517; andMimoto et al., Protein Engineering Design and Selection, vol. 26, No.10, pp. 589-598 (2013), each of which is hereby incorporated byreference in its entirety for all purposes, and in particular anyfigures, legends, or discussion related to variants that affect bindingto Fcγ receptors, including the FcγRIIb receptor. The combinationsdescribed in FIG. 48 may further include selections from additionaltarget antigens known in the art or described herein.

In still further embodiments and in accordance with any of the above,the rapid clearance molecules of the invention reduce the totalconcentration of free antigen in a patient as compared to theconcentration prior to treatment with the rapid clearance molecule. Inexemplary embodiments, methods and compositions of the invention reducethe total concentration of antigen by at least 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90-fold as compared to the concentration prior to treatmentwith the rapid clearance molecule.

In yet further embodiments and in accordance with any of the above,rapid clearance molecules include amino acid substitutions (includingthose described herein) that lead to Fc variants with increased FcγRIIbas compared to the parent Fc domain and further also alates binding toFcγRIIa. Such Fc variants may include the Fc variants described hereinas well as Fc variants described herein combined with furthersubstitutions that ablate binding to other FcγR, including withoutlimitation FcγRIIa. As discussed above, “ablation” herein is meant adecrease or removal of activity. Thus for example, “ablating FcγRbinding” means the Fc region amino acid variant has less than 50%starting binding as compared to an Fc region not containing the specificvariant, with less than 70-80-90-95-98% loss of activity beingpreferred, and in general, with the activity being below the level ofdetectable binding in a Biacore assay.

In certain aspects and in accordance with any of the above, acceleratedclearance of the antigen containing complexes seen with rapid clearancemolecules containing amino acid substitutions that confer high affinity(as compared to the parent Fc domain) to the inhibitory receptor FcγRIIbis likely mediated by interaction with FcγRIIb-expressing cells,possibly liver sinusoidal endothelial cells. In certain embodiments, theaccelerated clearance of the antigen containing molecules is notmediated by changes in pH or ionic conditions, such as those encounteredwithin lysosomes.

In general, antibodies of the invention that have engineered Fc domainsthat result in higher affinity than wild-type antibodies to the FcγRIIbreceptor can be directed to a variety of antigens as discussed herein,including cancer antigens, pathogen antigens, allergy antigens, etc.

In further embodiments, rapid clearance antibodies of the invention showfunctional properties as described in U.S. Provisional Application Ser.No. 61/752,955, filed Jan. 15, 2013; 61/794,164, filed Mar. 15, 2013,61/794,386, filed Mar. 15, 2013, and 61/833,696, filed Jun. 11, 2013,each of which is expressly incorporated by reference in the entirety andin particular the figures describing such variants, their functionalproperties, or models of their functional properties (as shown forexample in FIGS. 23-26 of U.S. Ser. No. 61/752,955).

Antibodies

The present invention relates to the generation of heterodimericantibodies, generally therapeutic antibodies, through the use of“heterodimerization amino acid variants”. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1, CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown herein, thepresent invention covers heterodimers that can contain one or bothchains that are IgG1/G2 hybrids (see SEQ ID NO:6, for example).

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546, entirely incorporated byreference) which consists of a single variable, (v) isolated CDRregions, (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)bispecific single chain Fv (WO 03/11161, hereby incorporated byreference) and (ix) “diabodies” or “triabodies”, multivalent ormultispecific fragments constructed by gene fusion (Tomlinson et. al.,2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993,Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated byreference). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245, entirely incorporated by reference).

Chimeric and Humanized Antibodies

In some embodiments, the scaffold components can be a mixture fromdifferent species. As such, if the protein is an antibody, such antibodymay be a chimeric antibody and/or a humanized antibody. In general, both“chimeric antibodies” and “humanized antibodies” refer to antibodiesthat combine regions from more than one species. For example, “chimericantibodies” traditionally comprise variable region(s) from a mouse (orrat, in some cases) and the constant region(s) from a human. “Humanizedantibodies” generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In some cases, the scFv can be joined to the Fc region, and may includesome or the entire hinge region.

Fc Fusion Proteins

In addition to antibody constructs discussed herein, the inventionfurther provides Fc fusion proteins where the Fc region has IIbvariants. That is, rather than have the Fc domain of an antibody joinedto an antibody variable region, the Fc domain can be joined to othermoieties, particularly binding moieties such as ligands. By “Fc fusion”as used herein is meant a protein wherein one or more polypeptides isoperably linked to an Fc region. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) as used in the prior art(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200, both entirely incorporated byreference). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general can be any protein or smallmolecule. Virtually any protein or small molecule may be linked to Fc togenerate an Fc fusion. Protein fusion partners may include, but are notlimited to, the variable region of any antibody, the target-bindingregion of a receptor, an adhesion molecule, a ligand, an enzyme, acytokine, a chemokine, or some other protein or protein domain. Smallmolecule fusion partners may include any therapeutic agent that directsthe Fc fusion to a therapeutic target. Such targets may be any molecule,preferably an extracellular receptor, which is implicated in disease.Thus, the IgG variants can be linked to one or more fusion partners.

FcγRIIb Variants

The compositions and methods of the invention rely on FcγRIIb variantsthat increase binding to the FcγRIIb receptor. Related applicationsdiscuss the FcγRIIb variants in detail. See for example U.S. Ser. Nos.11/124,620 and 13/294,103, both of which are incorporated by referencein their entirety, and in particular for the amino acid variantpositions, accompanying specification description, figures andaccompanying legends, and data relating to the variants. Fc variantsthat find particular use herein include, but are not limited to, thoselisted in FIG. 30.

FcγRIIb receptor variants that are considered “tight” binding anddisplay the fastest rapid clearance times, include S267E/L328F.

FcγRIIb receptor variants increased binding as compared to wild type Fcdomains, e.g. IgG1 domains, but are considered lower affinity and thuscan result in longer half lives include, but are not limited to, S267Eand G236N/267E and G236D/267E (sometimes referred to as “IIb-lite”variants).

Additional FcγR Variants

In addition to FcγRIIb receptor variants, there are a number of usefulFc substitutions that can be made to alter binding to one or more of theFcγR receptors. Substitutions that result in increased binding as wellas decreased binding can be useful. For example, it is known thatincreased binding to FcγRIIIa generally results in increased ADCC(antibody dependent cell-mediated cytotoxicity; the cell-mediatedreaction wherein nonspecific cytotoxic cells that express FcγRsrecognize bound antibody on a target cell and subsequently cause lysisof the target cell. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), U.S. Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, S267E, L328F, S267E/L328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243L, 298A and 299T. In somecases ablation variants such as 236R, 328R, and 236R/328R can be made,although this is not preferred in some embodiments. Additional suitableFc variants are found in FIG. 41 of US 2006/0024298, the figure andlegend of which are hereby incorporated by reference in their entirety.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and optionally increased serumhalf life, as specifically disclosed in U.S. Ser. No. 12/341,769, herebyincorporated by reference in its entirety, including, but not limitedto, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436Ior V/434S, 436V/428L and 259I/308F/428L.

IgE-FcγRIIb Molecules

One example of rapid clearance applications are antibodies that containan antigen binding site to IgE and FcγRIIb variants, resulting in“co-engagement”. For example, described herein are variant anti-IgEantibodies engineered such that the Fc domain binds to FcγRIIb with upto ˜430-fold greater affinity relative to native IgG1. These FcγRIIbbinding-enhanced (IIbE) variants strongly inhibit BCR-induced calciummobilization and viability in primary human IgE+ B cells. The use of asingle molecule, such as an antibody to suppress B cell functions ofcognate IgE BCR and FcγRIIb represents a novel approach in the treatmentof IgE-mediated diseases. Non-limiting examples of IgE-mediated diseasesinclude allergic responses and asthma and are described in U.S. Ser. No.12/156,183, hereby incorporated by reference in its entirety, andparticularly for the discussion associated with coengagement, anddescribed below.

Factor VIII-FcγRIIb Molecules

In one embodiment, the rapid clearance mechanisms of the presentinvention are used to treat hemophilia. One issue with hemophiliacs isthe effect that Factor VIII (FVIII (not to be confused with “Fv”))inhibitors play in disease. The most significant complication oftreatment of hemophilia A is the development of alloantibodies to thetherapeutic Factor VIII, that then inhibit the activity of the FactorVIII. Approximately 30% of patients with severe hemophilia A developthese alloantibodies, recognizing the exogenous correct FVIII as“foreign”, generally resulting in bleeding episodes that are moredifficult to manage.

FIG. 28 shows the structure of the FVIIIa protein, consisting ofheterodimeric protein comprising a heavy chain (A1-A2-B, not to beconfused with the heavy chain of an antibody) and a light chain(A3-C1-C2) that are associated through a noncovalent divalent metal ionlinkage between the A1 and A3 domains. The alloantibodies generallydevelop to the A2 and C2 domains, which are the dominant epitopes forthe alloantibodies, generally accounting for roughly 68% of thealloantibody antigens.

Due to the rapid clearance of antibodies and Fc fusions engineered tobind with high affinity to FcγRIIb, the present invention provides“scrubber” or “drug” Fc fusions, comprising some or all of the domainsof FVIIIa, that bind to the FVIIIa inhibitors thereby clearing out thealloantibodies. That is, the FcγRIIb fusions, directed against theseinhibitor antibodies will sequester the inhibitor antibodies, rapidlyclear the inhibitor antibodies, and will also inhibit FVIII-reactive Bcells (to prohibit the further production of the inhibitors).

CR-Fc Fusion Molecules

In one embodiment, the rapid clearance mechanisms of the presentinvention are used with complement receptor 2 (CR2)-Fc fusions forimmune system modulation and accelerated clearance of C3d-tagged immunecomplexes. These fusion proteins in this context are sometimes referredto herein as “CR2-IIbE”. In this embodiment, increased affinity for theinhibitory Fc receptor FcRIIb (CD32b) is utilized to facilitate rapid invivo clearance of C3-tagged immune complexes via their interaction withCR-IIbE fusions. Incorporation of the IIb-enhancing affinitysubstitutions into various fusions leads to a novel phenomenon wherebythe fusion-target complex is cleared extremely rapidly while the CR-IIbEalone retains a reasonably long half-life. Application of differentIIb-enhancing substitutions (e.g. S267E/L328F, G236D/S267E, 236N/267E,or S267E alone, as are useful for all the rapid clearance moleculesherein) may lead to different balances between the complex clearancerate and the fusion protein clearance rate, allowing for tailoringtoward optimal therapeutic profile and dosing.

In another aspect of the invention, rapid clearance-mediating IIbtechnology can additionally be applied to other complement systemreceptors or inhibitors, including but not limited to, CR1, Factor H(fH), CR3, and CRIg. Typically, only the SCR domains required forrecognition of the appropriate complement factor will be required,although additional repeats may be included for stability.

In yet another aspect of the invention, rapid clearance-mediating IIbtechnology can be applied to antibodies that recognize C3 fragments,thereby mimicking the above-described CR-Fc fusions, similar to the IgEand Factor VIII antibody examples. Examples include, but are not limitedto, anti-C3d antibodies with engineered Fc regions. See also U.S. Ser.No. 61/752,955, filed Jan. 15, 2013, which is hereby incorporated byreference in its entirety for all purposes and in particular for allteachings, figures and legends related to schemes of complement andantibodies directed to same.

Anti-IgE Antibodies

In some embodiments, the immunoglobulins described herein bind IgE. Theanti-IgE antibodies of the invention may comprise any variable region,known or not yet known, that has specificity for IgE. Known anti-IgEantibodies include but are not limited to murine antibodies MaE11,MaE13, and MaE15, humanized and/or engineered versions of theseantibodies including E25, E26, and E27, particularly E25, also known asrhuMab-E25, also known as Omalizumab, such as those described in U.S.Pat. No. 6,761,889, U.S. Pat. No. 6,329,509, US20080003218A1, andPresta, L G et al., 1993, J Immunol 151:2623-2632, all herein expresslyincorporated by reference. A preferred engineered version of MaE11 is H1L1 MaE11, described in the Examples herein. Other anti-IgE that may beuseful for the invention include murine antibody TES-C21, chimericTES-C21, also known as CGP51901 (Corne, J et al., 1997, J Clin Invest99:879-887; Racine-Poon, A et al., 1997, Clin Pharmcol Ther 62:675-690),and humanized and/or engineered versions of this antibody including butnot limited to CGP56901, also known as TNX-901, such as those antibodiesdescribed in Kolbinger, F et al., 1993, Protein Eng 6:971-980. Otheranti-IgE antibodies that may find use for the invention are described inU.S. Pat. No. 6,066,718, U.S. Pat. No. 6,072,035, PCT/US04/02894, U.S.Pat. No. 5,342,924, U.S. Pat. No. 5,091,313, U.S. Pat. No. 5,449,760,U.S. Pat. No. 5,543,144, U.S. Pat. No. 5,342,924, and U.S. Pat. No.5,614,611, all of which are incorporated herein by reference. Otheruseful anti-IgE antibodies include the murine antibody BSW17. Amino acidsequences of the variable region VH and VL domains and CDRs of some ofthese antibodies are provided in FIG. 5.

Fc Variants and Fc Receptor Binding Properties

Immunoglobulins disclosed herein may comprise an Fc variant. An Fcvariant comprises one or more amino acid modifications relative to aparent Fc polypeptide, wherein the amino acid modification(s) provideone or more optimized properties. An Fc variant disclosed herein differsin amino acid sequence from its parent by virtue of at least one aminoacid modification. Thus Fc variants disclosed herein have at least oneamino acid modification compared to the parent. Alternatively, the Fcvariants disclosed herein may have more than one amino acid modificationas compared to the parent, for example from about one to fifty aminoacid modifications, e.g., from about one to ten amino acidmodifications, from about one to about five amino acid modifications,etc. compared to the parent. Thus the sequences of the Fc variants andthose of the parent Fc polypeptide are substantially homologous. Forexample, the variant Fc variant sequences herein will possess about 80%homology with the parent Fc variant sequence, e.g., at least about 90%homology, at least about 95% homology, at least about 98% homology, atleast about 99% homology, etc. Modifications disclosed herein includeamino acid modifications, including insertions, deletions, andsubstitutions. Modifications disclosed herein also include glycoformmodifications. Modifications may be made genetically using molecularbiology, or may be made enzymatically or chemically.

Fc variants disclosed herein are defined according to the amino acidmodifications that compose them. Thus, for example, S267E is an Fcvariant with the substitution S267E relative to the parent Fcpolypeptide. Likewise, S267E/L328F defines an Fc variant with thesubstitutions S267E and L328F relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as S267E/L328F. It is noted thatthe order in which substitutions are provided is arbitrary, that is tosay that, for example, S267E/L328F is the same Fc variant as L328F/267E,and so on. Unless otherwise noted, positions discussed herein arenumbered according to the EU index or EU numbering scheme (Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed., UnitedStates Public Health Service, National Institutes of Health, Bethesda,hereby entirely incorporated by reference). The EU index or EU index asin Kabat or EU numbering scheme refers to the numbering of the EUantibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, herebyentirely incorporated by reference).

In certain embodiments, the Fc variants disclosed herein are based onhuman IgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared, including but notlimited to sequences from other organisms, for example rodent andprimate sequences. Immunoglobulins may also comprise sequences fromother immunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like.It is contemplated that, although the Fc variants disclosed herein areengineered in the context of one parent IgG, the variants may beengineered in or “transferred” to the context of another, second parentIgG. This is done by determining the “equivalent” or “corresponding”residues and substitutions between the first and second IgG, typicallybased on sequence or structural homology between the sequences of thefirst and second IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore of the homology alignment programs known in the art (for exampleusing conserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first immunoglobulin are defined. Alignment ofconserved residues may conserve 100% of such residues. However,alignment of greater than 75% or as little as 50% of conserved residuesis also adequate to define equivalent residues. Equivalent residues mayalso be defined by determining structural homology between a first andsecond IgG that is at the level of tertiary structure for IgGs whosestructures have been determined. In this case, equivalent residues aredefined as those for which the atomic coordinates of two or more of themain chain atoms of a particular amino acid residue of the parent orprecursor (N on N, CA on CA, C on C and O on O) are within about 0.13nm, after alignment. In another embodiment, equivalent residues arewithin about 0.1 nm after alignment. Alignment is achieved after thebest model has been oriented and positioned to give the maximum overlapof atomic coordinates of non-hydrogen protein atoms of the proteins.Regardless of how equivalent or corresponding residues are determined,and regardless of the identity of the parent IgG in which the IgGs aremade, what is meant to be conveyed is that the Fc variants discovered asdisclosed herein may be engineered into any second parent IgG that hassignificant sequence or structural homology with the Fc variant. Thusfor example, if a variant antibody is generated wherein the parentantibody is human IgG1, by using the methods described above or othermethods for determining equivalent residues, the variant antibody may beengineered in another IgG1 parent antibody that binds a differentantigen, a human IgG2 parent antibody, a human IgA parent antibody, amouse IgG2a or IgG2b parent antibody, and the like. Again, as describedabove, the context of the parent Fc variant does not affect the abilityto transfer the Fc variants disclosed herein to other parent IgGs.

The Fc variants disclosed herein may be optimized for a variety of Fcreceptor binding properties. An Fc variant that is engineered orpredicted to display one or more optimized properties is herein referredto as an “optimized Fc variant”. Properties that may be optimizedinclude but are not limited to enhanced or reduced affinity for an FcγR.In one embodiment, the Fc variants disclosed herein are optimized topossess enhanced affinity for an inhibitory receptor FcγRIIb. In otherembodiments, immunoglobulins disclosed herein provide enhanced affinityfor FcγRIIb, yet reduced affinity for one or more activating FcγRs,including for example FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. TheFcγR receptors may be expressed on cells from any organism, includingbut not limited to human, cynomolgus monkeys, and mice. The Fc variantsdisclosed herein may be optimized to possess enhanced affinity for humanFcγRIIb.

By “greater affinity” or “improved affinity” or “enhanced affinity” or“better affinity” than a parent Fc polypeptide, as used herein is meantthat an Fc variant binds to an Fc receptor with a significantly higherequilibrium constant of association (KA or Ka) or lower equilibriumconstant of dissociation (KD or Kd) than the parent Fc polypeptide whenthe amounts of variant and parent polypeptide in the binding assay areessentially the same. For example, the Fc variant with improved Fcreceptor binding affinity may display from about 5 fold to about 1000fold, e.g. from about 10 fold to about 500 fold improvement in Fcreceptor binding affinity compared to the parent Fc polypeptide, whereFc receptor binding affinity is determined, for example, by the bindingmethods disclosed herein, including but not limited to Biacore, by oneskilled in the art. Accordingly, by “reduced affinity” as compared to aparent Fc polypeptide as used herein is meant that an Fc variant bindsan Fc receptor with significantly lower KA or higher KD than the parentFc polypeptide. Greater or reduced affinity can also be defined relativeto an absolute level of affinity. For example, according to the dataherein, WT (native) IgG1 binds FcγRIIb with an affinity of about 2 μM,or about 2000 nM. Furthermore, some Fc variants described herein bindFcγRIIb with an affinity about 10-fold greater to WT IgG1. As disclosedherein, greater or enhanced affinity means having a KD lower than about100 nM, for example between about 10 nM-about 100 nM, between about1-about 100 nM, or less than about 1 nM.

Anti-IgE antibodies of the invention preferably have high affinity forFcγRIIb. By high affinity herein is meant that the affinity of theinteraction between the antibody and FcγRIIb is stronger than 100 nM.That is to say that the equilibrium dissociation constant Kd for bindingof the antibody to FcγRIIb is lower than 100 nM.

In one embodiment, the Fc variants provide selectively enhanced affinityto FcγRIIb relative to one or more activating receptors. Selectivelyenhanced affinity means either that the Fc variant has improved affinityfor FcγRIIb relative to the activating receptor(s) as compared to theparent Fc polypeptide but has reduced affinity for the activatingreceptor(s) as compared to the parent Fc polypeptide, or it means thatthe Fc variant has improved affinity for both FcγRIIb and activatingreceptor(s) as compared to the parent Fc polypeptide, however theimprovement in affinity is greater for FcγRIIb than it is for theactivating receptor(s). In alternate embodiments, the Fc variants reduceor ablate binding to one or more activating FcγRs, reduce or ablatebinding to one or more complement proteins, reduce or ablate one or moreFcγR-mediated effector functions, and/or reduce or ablate one or morecomplement-mediated effector functions.

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the Fcvariants disclosed herein. Whereas the specificity and selectivity of agiven Fc variant for the different classes of FcγRs significantlyaffects the capacity of an Fc variant to target a given antigen fortreatment of a given disease, the specificity or selectivity of an Fcvariant for different polymorphic forms of these receptors may in partdetermine which research or pre-clinical experiments may be appropriatefor testing, and ultimately which patient populations may or may notrespond to treatment. Thus the specificity or selectivity of Fc variantsdisclosed herein to Fc receptor polymorphisms, including but not limitedto FcγRIIa, FcγRIIIa, and the like, may be used to guide the selectionof valid research and pre-clinical experiments, clinical trial design,patient selection, dosing dependence, and/or other aspects concerningclinical trials.

Fc variants disclosed herein may comprise modifications that modulateinteraction with Fc receptors other than FcγRs, including but notlimited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).FcRHs include but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5,and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

An important parameter that determines the most beneficial selectivityof a given Fc variant to treat a given disease is the context of the Fcvariant. Thus the Fc receptor selectivity or specificity of a given Fcvariant will provide different properties depending on whether itcomposes an antibody, Fc fusion, or Fc variants with a coupled fusionpartner. In one embodiment, an Fc receptor specificity of the Fc variantdisclosed herein will determine its therapeutic utility. The utility ofa given Fc variant for therapeutic purposes will depend on the epitopeor form of the target antigen and the disease or indication beingtreated. For some targets and indications, greater FcγRIIb affinity andreduced activating FcγR-mediated effector functions may be beneficial.For other target antigens and therapeutic applications, it may bebeneficial to increase affinity for FcγRIIb, or increase affinity forboth FcγRIIb and activating receptors.

Glycoform Modifications

Many polypeptides, including antibodies, are subjected to a variety ofposttranslational modifications involving carbohydrate moieties, such asglycosylation with oligosaccharides. There are several factors that caninfluence glycosylation. The species, tissue and cell type have all beenshown to be important in the way that glycosylation occurs. In addition,the extracellular environment, through altered culture conditions suchas serum concentration, may have a direct effect on glycosylation(Lifely et al., 1995, Glycobiology 5(8): 813-822).

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate (Jefferiset al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, TrendsBiotech 15:26-32). For human IgG, the core oligosaccharide normallyconsists of GlcNAc2Man3GlcNAc, with differing numbers of outer residues.

The carbohydrate moieties of immunoglobulins disclosed herein will bedescribed with reference to commonly used nomenclature for thedescription of oligosaccharides. A review of carbohydrate chemistrywhich uses this nomenclature is found in Hubbard et al. 1981, Ann. Rev.Biochem. 50:555-583. This nomenclature includes, for instance, Man,which represents mannose; GlcNAc, which represents2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucose;and Glc, which represents glucose. Sialic acids are described by theshorthand notation NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or O-linkages. Theoligosaccharides of immunoglobulins disclosed herein occur generally areattached to a CH2 domain of an Fc region as N-linked oligosaccharides.“N-linked glycosylation” refers to the attachment of the carbohydratemoiety to an asparagine residue in a glycoprotein chain. The skilledartisan will recognize that, for example, each of murine IgG1, IgG2a,IgG2b and IgG3 as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2domains have a single site for N-linked glycosylation at amino acidresidue 297 (Kabat et al. Sequences of Proteins of ImmunologicalInterest, 1991).

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)2 typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the β1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a β(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

Described herein are Fc variants that comprise modified glycoforms orengineered glycoforms. By “modified glycoform” or “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Engineered glycoforms may be useful for a variety of purposes,including but not limited to enhancing or reducing FcγR-mediatedeffector function. In one embodiment, the immunoglobulins disclosedherein are modified to control the level of fucosylated and/or bisectingoligosaccharides that are covalently attached to the Fc region.

A variety of methods are well known in the art for generating modifiedglycoforms (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies etal., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCTWO 02/30954A1); (Potelligent™ technology [Biowa, Inc., Princeton, N.J.];GlycoMAb™ glycosylation engineering technology [GLYCART biotechnologyAG, Zürich, Switzerland]; all of which are expressly incorporated byreference). These techniques control the level of fucosylated and/orbisecting oligosaccharides that are covalently attached to the Fcregion, for example by expressing an IgG in various organisms or celllines, engineered or otherwise (for example Lec-13 CHO cells or rathybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/orβ1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the IgG has been expressed. Other methods formodifying glycoforms of the immunoglobulins disclosed herein includeusing glycoengineered strains of yeast (Li et al., 2006, NatureBiotechnology 24(2):210-215), moss (Nechansky et al., 2007, Mol Immunjol44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). The use of a particular method to generate a modifiedglycoform is not meant to constrain embodiments to that method. Rather,embodiments disclosed herein encompass Fc variants with modifiedglycoforms irrespective of how they are produced.

In one embodiment, immunoglobulins disclosed herein are glycoengineeredto alter the level of sialylation. Higher levels of sialylated Fcglycans in immunoglobulin G molecules can adversely impact functionality(Scallon et al., 2007, Mol Immunol. 44(7):1524-34), and differences inlevels of Fc sialylation can result in modified anti-inflammatoryactivity (Kaneko et al., 2006, Science 313:670-673). Because antibodiesmay acquire anti-inflammatory properties upon sialylation of Fc corepolysaccharide, it may be advantageous to glycoengineer theimmunoglobulins disclosed herein for greater or reduced Fc sialic acidcontent.

Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus for example an immunoglobulin may comprise anengineered glycoform. Alternatively, engineered glycoform may refer tothe immunoglobulin that comprises the different carbohydrate oroligosaccharide. In one embodiment, a composition disclosed hereincomprises a glycosylated Fc variant having an Fc region, wherein about51-100% of the glycosylated antibody, e.g., 80-100%, 90-100%, 95-100%,etc. of the antibody in the composition comprises a mature corecarbohydrate structure which lacks fucose. In another embodiment, theantibody in the composition both comprises a mature core carbohydratestructure that lacks fucose and additionally comprises at least oneamino acid modification in the Fc region. In an alternative embodiment,a composition comprises a glycosylated Fc variant having an Fc region,wherein about 51-100% of the glycosylated antibody, 80-100%, or 90-100%,of the antibody in the composition comprises a mature core carbohydratestructure which lacks sialic acid. In another embodiment, the antibodyin the composition both comprises a mature core carbohydrate structurethat lacks sialic acid and additionally comprises at least one aminoacid modification in the Fc region. In yet another embodiment, acomposition comprises a glycosylated Fc variant having an Fc region,wherein about 51-100% of the glycosylated antibody, 80-100%, or 90-100%,of the antibody in the composition comprises a mature core carbohydratestructure which contains sialic acid. In another embodiment, theantibody in the composition both comprises a mature core carbohydratestructure that contains sialic acid and additionally comprises at leastone amino acid modification in the Fc region. In another embodiment, thecombination of engineered glycoform and amino acid modification providesoptimal Fc receptor binding properties to the antibody.

Other Modifications

Immunoglobulins disclosed herein may comprise one or more modificationsthat provide optimized properties that are not specifically related toFcγR- or complement-mediated effector functions per se. Saidmodifications may be amino acid modifications, or may be modificationsthat are made enzymatically or chemically. Such modification(s) likelyprovide some improvement in the immunoglobulin, for example anenhancement in its stability, solubility, function, or clinical use.Disclosed herein are a variety of improvements that may be made bycoupling the immunoglobulins disclosed herein with additionalmodifications.

In one embodiment, the variable region of an antibody disclosed hereinmay be affinity matured, that is to say that amino acid modificationshave been made in the VH and/or VL domains of the antibody to enhancebinding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the targetantigen. Immunoglobulins disclosed herein may comprise one or moremodifications that provide reduced or enhanced internalization of animmunoglobulin.

In one embodiment, modifications are made to improve biophysicalproperties of the immunoglobulins disclosed herein, including but notlimited to stability, solubility, and oligomeric state. Modificationscan include, for example, substitutions that provide more favorableintramolecular interactions in the immunoglobulin such as to providegreater stability, or substitution of exposed nonpolar amino acids withpolar amino acids for higher solubility. Other modifications to theimmunoglobulins disclosed herein include those that enable the specificformation or homodimeric or homomultimeric molecules. Such modificationsinclude but are not limited to engineered disulfides, as well aschemical modifications or aggregation methods which may provide amechanism for generating covalent homodimeric or homomultimers.Additional modifications to the variants disclosed herein include thosethat enable the specific formation or heterodimeric, heteromultimeric,bifunctional, and/or multifunctional molecules. Such modificationsinclude, but are not limited to, one or more amino acid substitutions inthe CH3 domain, in which the substitutions reduce homodimer formationand increase heterodimer formation. Additional modifications includemodifications in the hinge and CH3 domains, in which the modificationsreduce the propensity to form dimers.

In further embodiments, the immunoglobulins disclosed herein comprisemodifications that remove proteolytic degradation sites. These mayinclude, for example, protease sites that reduce production yields, aswell as protease sites that degrade the administered protein in vivo. Inone embodiment, additional modifications are made to remove covalentdegradation sites such as deamidation (i.e. deamidation of glutaminyland asparaginyl residues to the corresponding glutamyl and aspartylresidues), oxidation, and proteolytic degradation sites. Deamidationsites that are particular useful to remove are those that have enhancepropensity for deamidation, including, but not limited to asparaginyland glutamyl residues followed by glycines (NG and QG motifs,respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), incorporated entirely by reference), acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.Additional modifications also may include but are not limited toposttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The immunoglobulins disclosed herein may comprise modifications thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci.U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin etal., 2003, Science 301(5635):964-7, all incorporated entirely byreference. In some embodiments, these modifications enable manipulationof various functional, biophysical, immunological, or manufacturingproperties discussed above. In additional embodiments, thesemodifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theimmunoglobulin may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. Additional amino acid modifications may be made to enablespecific or non-specific chemical or posttranslational modification ofthe immunoglobulins. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the immunoglobulins disclosed herein.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an antibodydisclosed herein. See for example U.S. Ser. No. 09/903,378, U.S. Ser.No. 10/754,296, U.S. Ser. No. 11/249,692, and references cited therein,all expressly incorporated by reference.

In some embodiments, immunoglobulins disclosed herein may be combinedwith immunoglobulins that alter FcRn binding. Such variants may provideimproved pharmacokinetic properties to the immunoglobulins. Preferredvariants that increase binding to FcRn and/or improve pharmacokineticproperties include but are not limited to substitutions at positions259, 308, 428, and 434, including but not limited to for example 259I,308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M (PCT/US2008/088053,filed Dec. 22, 2008, entitled “Fc Variants with Altered Binding toFcRn”, entirely incorporated by reference). Other variants that increaseFc binding to FcRn include but are not limited to: 250E, 250Q, 428L,428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216,Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A,305A, 307A, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al,Journal of Biological Chemistry, 2001, 276(9):6591-6604, entirelyincorporated by reference), 252F, 252T, 252Y, 252W, 254T, 256S, 256R,256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H,434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311 S (Dall Acquaet al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al.,2006, The Journal of biological chemistry 281:23514-23524, entirelyincorporated by reference).

Covalent modifications of antibodies are included within the scope ofimmunoglobulins disclosed herein, and are generally, but not always,done post-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

In some embodiments, the covalent modification of the antibodiesdisclosed herein comprises the addition of one or more labels. The term“labeling group” means any detectable label. In some embodiments, thelabeling group is coupled to the antibody via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in generatingimmunoglobulins disclosed herein.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, U.S. Pat. Nos. 7,303,749, 7,601,354, U.S. Ser. No.12/631,508, WO02/098883, U.S. Pat. Nos. 6,441,163, 7,368,565, WO02/16368and WO04/1033272, all of which are expressly incorporated by referencein their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,U.S. Pat. Nos. 5,767,237 and 6,124,431, expressly incorporated byreference in their entirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by reference) andduocarmycins are members of a family of antitumor antibiotics utilizedin ADCs. These antibiotics appear to work through sequence-selectivelyalkylating DNA at the N3 of adenine in the minor groove, which initiatesa cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, U.S. Pat. Nos. 5,703,080, 6,989,452, 7,087,600,7,129,261, 7,498,302, and 7,507,420, all of which are expresslyincorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with amultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 96)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Conjugates

In one embodiment, the molecules disclosed herein are antibody “fusionproteins”, sometimes referred to herein as “antibody conjugates”. Thefusion partner or conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody and on the conjugate partner. Conjugate andfusion partners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1999, Curr. Opin.Immunol. 11:584-588, incorporated entirely by reference. Possibleconjugate partners include but are not limited to cytokines, cytotoxicagents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenicagents, a tyrosine kinase inhibitors, and other therapeutically activeagents. In some embodiments, conjugate partners may be thought of moreas payloads, that is to say that the goal of a conjugate is targeteddelivery of the conjugate partner to a targeted cell, for example acancer cell or immune cell, by the immunoglobulin. Thus, for example,the conjugation of a toxin to an immunoglobulin targets the delivery ofsaid toxin to cells expressing the target antigen. As will beappreciated by one skilled in the art, in reality the concepts anddefinitions of fusion and conjugate are overlapping. The designation ofa fusion or conjugate is not meant to constrain it to any particularembodiment disclosed herein. Rather, these terms are used loosely toconvey the broad concept that any immunoglobulin disclosed herein may belinked genetically, chemically, or otherwise, to one or morepolypeptides or molecules to provide some desirable property.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diphtheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs.

In one embodiment, the molecules disclosed herein are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J. Immunol. Methods 248:91-101, incorporatedentirely by reference, cytokines may be fused to antibody to provide anarray of desirable properties. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; C5a; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In yet another embodiment, an molecules disclosed herein may beconjugated to a “receptor” (such streptavidin) for utilization in tumorpretargeting wherein the immunoglobulin-receptor conjugate isadministered to the patient, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the immunoglobulin isconjugated or operably linked to an enzyme in order to employ AntibodyDependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the immunoglobulin to aprodrug-activating enzyme that converts a prodrug (e.g. a peptidylchemotherapeutic agent.

When immunoglobulin partners are used as conjugates, conjugate partnersmay be linked to any region of an immunoglobulin disclosed herein,including at the N- or C-termini, or at some residue in-between thetermini. A variety of linkers may find use in immunoglobulins disclosedherein to covalently link conjugate partners to an immunoglobulin. By“linker”, “linker sequence”, “spacer”, “tethering sequence” orgrammatical equivalents thereof, herein is meant a molecule or group ofmolecules (such as a monomer or polymer) that connects two molecules andoften serves to place the two molecules in one configuration. Linkersare known in the art; for example, homo- or hetero-bifunctional linkersas are well known (see, 1994 Pierce Chemical Company catalog, technicalsection on cross-linkers, pages 155-200, incorporated entirely byreference). A number of strategies may be used to covalently linkmolecules together. These include, but are not limited to polypeptidelinkages between N- and C-termini of proteins or protein domains,linkage via disulfide bonds, and linkage via chemical cross-linkingreagents. In one aspect of this embodiment, the linker is a peptidebond, generated by recombinant techniques or peptide synthesis. Thelinker peptide may predominantly include the following amino acidresidues: Gly, Ser, Ala, or Thr. The linker peptide should have a lengththat is adequate to link two molecules in such a way that they assumethe correct conformation relative to one another so that they retain thedesired activity. Suitable lengths for this purpose include at least oneand not more than 50 amino acid residues. In one embodiment, the linkeris from about 1 to 30 amino acids in length, e.g., a linker may be 1 to20 amino acids in length. Useful linkers include glycine-serine polymers(including, for example, (GS)n, (GSGGS)n (Set forth as SEQ ID NO:1),(GGGGS)n (Set forth as SEQ ID NO:2) and (GGGS)n (Set forth as SEQ IDNO:3), where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers, and other flexible linkers, as will beappreciated by those in the art. Alternatively, a variety ofnonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers.

Production

Also disclosed herein are methods for producing and experimentallytesting the antibodies used in the methods described herein. Thedisclosed methods are not meant to constrain embodiments to anyparticular application or theory of operation. Rather, the providedmethods are meant to illustrate generally that one or moreimmunoglobulins may be produced and experimentally tested to obtainimmunoglobulins. General methods for antibody molecular biology,expression, purification, and screening are described in AntibodyEngineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689;Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: ALaboratory Manual by Harlow & Lane, New York: Cold Spring HarborLaboratory Press, 1988, all incorporated entirely by reference.

In one embodiment disclosed herein, nucleic acids are created thatencode the molecules, and that may then be cloned into host cells,expressed and assayed, if desired. Thus, nucleic acids, and particularlyDNA, may be made that encode each protein sequence. These practices arecarried out using well-known procedures. For example, a variety ofmethods that may find use in generating immunoglobulins disclosed hereinare described in Molecular Cloning—A Laboratory Manual, 3rd Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons), bothincorporated entirely by reference. As will be appreciated by thoseskilled in the art, the generation of exact sequences for a librarycomprising a large number of sequences is potentially expensive and timeconsuming. By “library” herein is meant a set of variants in any form,including but not limited to a list of nucleic acid or amino acidsequences, a list of nucleic acid or amino acid substitutions atvariable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising thevariant proteins, either in purified or unpurified form. Accordingly,there are a variety of techniques that may be used to efficientlygenerate libraries disclosed herein. Such methods include but are notlimited to gene assembly methods, PCR-based method and methods which usevariations of PCR, ligase chain reaction-based methods, pooled oligomethods such as those used in synthetic shuffling, error-proneamplification methods and methods which use oligos with randommutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in for generating nucleic acidsthat encode immunoglobulins.

The molecules disclosed herein may be produced by culturing a host celltransformed with nucleic acid, e.g., an expression vector, containingnucleic acid encoding the molecules, under the appropriate conditions toinduce or cause expression of the protein. The conditions appropriatefor expression will vary with the choice of the expression vector andthe host cell, and will be easily ascertained by one skilled in the artthrough routine experimentation. A wide variety of appropriate hostcells may be used, including but not limited to mammalian cells,bacteria, insect cells, and yeast. For example, a variety of cell linesthat may find use in generating immunoglobulins disclosed herein aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In one embodiment, the molecules are expressed in mammalian expressionsystems, including systems in which the expression constructs areintroduced into the mammalian cells using virus such as retrovirus oradenovirus. Any mammalian cells may be used, e.g., human, mouse, rat,hamster, and primate cells. Suitable cells also include known researchcells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK,COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cells and variants thereof. In analternate embodiment, library proteins are expressed in bacterial cells.Bacterial expression systems are well known in the art, and includeEscherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris,and Streptococcus lividans. In alternate embodiments, immunoglobulinsare produced in insect cells (e.g. Sf21/Sf9, Trichoplusia niBti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia, etc). In analternate embodiment, molecules are expressed in vitro using cell freetranslation systems. In vitro translation systems derived from bothprokaryotic (e.g. E. coli) and eukaryotic (e.g. wheat germ, rabbitreticulocytes) cells are available and may be chosen based on theexpression levels and functional properties of the protein of interest.For example, as appreciated by those skilled in the art, in vitrotranslation is required for some display technologies, for exampleribosome display. In addition, the immunoglobulins may be produced bychemical synthesis methods. Also transgenic expression systems bothanimal (e.g. cow, sheep or goat milk, embryonated hen's eggs, wholeinsect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.)

The nucleic acids that encode the molecules disclosed herein may beincorporated into an expression vector in order to express the protein.A variety of expression vectors may be utilized for protein expression.Expression vectors may comprise self-replicating extra-chromosomalvectors or vectors which integrate into a host genome. Expressionvectors are constructed to be compatible with the host cell type. Thusexpression vectors which find use in generating immunoglobulinsdisclosed herein include but are not limited to those which enableprotein expression in mammalian cells, bacteria, insect cells, yeast,and in in vitro systems. As is known in the art, a variety of expressionvectors are available, commercially or otherwise, that may find use forexpressing molecules disclosed herein.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the molecule, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

molecules may be operably linked to a fusion partner to enable targetingof the expressed protein, purification, screening, display, and thelike. Fusion partners may be linked to the immunoglobulin sequence via alinker sequences. The linker sequence will generally comprise a smallnumber of amino acids, typically less than ten, although longer linkersmay also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs immunoglobulin and any associated fusion partners to adesired cellular location or to the extracellular media. As is known inthe art, certain signaling sequences may target a protein to be eithersecreted into the growth media, or into the periplasmic space, locatedbetween the inner and outer membrane of the cell. A fusion partner mayalso be a sequence that encodes a peptide or protein that enablespurification and/or screening. Such fusion partners include but are notlimited to polyhistidine tags (His-tags) (for example H6 and H10 orother tags for use with Immobilized Metal Affinity Chromatography (IMAC)systems (e.g. Ni+2 affinity columns)), GST fusions, MBP fusions,Strep-tag, the BSP biotinylation target sequence of the bacterial enzymeBirA, and epitope tags which are targeted by antibodies (for examplec-myc tags, flag-tags, and the like). As will be appreciated by thoseskilled in the art, such tags may be useful for purification, forscreening, or both. For example, an immunoglobulin may be purified usinga His-tag by immobilizing it to a Ni+2 affinity column, and then afterpurification the same His-tag may be used to immobilize the antibody toa Ni+2 coated plate to perform an ELISA or other binding assay (asdescribed below). A fusion partner may enable the use of a selectionmethod to screen immunoglobulins (see below). Fusion partners thatenable a variety of selection methods are well-known in the art. Forexample, by fusing the members of an immunoglobulin library to the geneIII protein, phage display can be employed (Kay et al., Phage display ofpeptides and proteins: a laboratory manual, Academic Press, San Diego,Calif., 1996; Lowman et al., 1991, Biochemistry 30:10832-10838; Smith,1985, Science 228:1315-1317, incorporated entirely by reference). Fusionpartners may enable immunoglobulins to be labeled. Alternatively, afusion partner may bind to a specific sequence on the expression vector,enabling the fusion partner and associated immunoglobulin to be linkedcovalently or noncovalently with the nucleic acid that encodes them. Themethods of introducing exogenous nucleic acid into host cells are wellknown in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In one embodiment, molecules are purified or isolated after expression.Proteins may be isolated or purified in a variety of ways known to thoseskilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use for purification ofimmunoglobulins disclosed herein. For example, the bacterial proteins Aand G bind to the Fc region. Likewise, the bacterial protein L binds tothe Fab region of some antibodies, as of course does the antibody'starget antigen. Purification can often be enabled by a particular fusionpartner. For example, immunoglobulins may be purified using glutathioneresin if a GST fusion is employed, Ni+2 affinity chromatography if aHis-tag is employed, or immobilized anti-flag antibody if a flag-tag isused. For general guidance in suitable purification techniques, see,e.g. incorporated entirely by reference Protein Purification: Principlesand Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994, incorporatedentirely by reference. The degree of purification necessary will varydepending on the screen or use of the immunoglobulins. In some instancesno purification is necessary. For example in one embodiment, if theimmunoglobulins are secreted, screening may take place directly from themedia. As is well known in the art, some methods of selection do notinvolve purification of proteins. Thus, for example, if a library ofimmunoglobulins is made into a phage display library, proteinpurification may not be performed.

In Vitro Experimentation

molecules may be screened using a variety of methods, including but notlimited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners has been discussed above. By “labeled” herein is meantthat the immunoglobulins disclosed herein have one or more elements,isotopes, or chemical compounds attached to enable the detection in ascreen. In general, labels fall into three classes: a) immune labels,which may be an epitope incorporated as a fusion partner that isrecognized by an antibody, b) isotopic labels, which may be radioactiveor heavy isotopes, and c) small molecule labels, which may includefluorescent and colorimetric dyes, or molecules such as biotin thatenable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In one embodiment, the functional and/or biophysical properties ofmolecules are screened in an in vitro assay. In vitro assays may allow abroad dynamic range for screening properties of interest. Propertiesthat may be screened include but are not limited to stability,solubility, and affinity for Fc ligands, for example FcγRs. Multipleproperties may be screened simultaneously or individually. Proteins maybe purified or unpurified, depending on the requirements of the assay.In one embodiment, the screen is a qualitative or quantitative bindingassay for binding of molecules to a protein or nonprotein molecule thatis known or thought to bind the molecule. In one embodiment, the screenis a binding assay for measuring binding to the target antigen. In analternate embodiment, the screen is an assay for binding of molecules toan Fc ligand, including but are not limited to the family of FcγRs, theneonatal receptor FcRn, the complement protein C1q, and the bacterialproteins A and G. Said Fc ligands may be from any organism. In oneembodiment, Fc ligands are from humans, mice, rats, rabbits, and/ormonkeys. Binding assays can be carried out using a variety of methodsknown in the art, including but not limited to FRET (FluorescenceResonance Energy Transfer) and BRET (Bioluminescence Resonance EnergyTransfer)-based assays, AlphaScreen™ (Amplified Luminescent ProximityHomogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBIACORE®), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the immunoglobulin. Assays may employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

The biophysical properties of molecules, for example stability andsolubility, may be tested using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, moleculesdisclosed herein may be unfolded using chemical denaturant, heat, or pH,and this transition may be monitored using methods including but notlimited to circular dichroism spectroscopy, fluorescence spectroscopy,absorbance spectroscopy, NMR spectroscopy, calorimetry, and proteolysis.As will be appreciated by those skilled in the art, the kineticparameters of the folding and unfolding transitions may also bemonitored using these and other techniques. The solubility and overallstructural integrity of an molecule may be quantitatively orqualitatively determined using a wide range of methods that are known inthe art. Methods which may find use for characterizing the biophysicalproperties of molecules disclosed herein include gel electrophoresis,isoelectric focusing, capillary electrophoresis, chromatography such assize exclusion chromatography, ion-exchange chromatography, andreversed-phase high performance liquid chromatography, peptide mapping,oligosaccharide mapping, mass spectrometry, ultraviolet absorbancespectroscopy, fluorescence spectroscopy, circular dichroismspectroscopy, isothermal titration calorimetry, differential scanningcalorimetry, analytical ultra-centrifugation, dynamic light scattering,proteolysis, and cross-linking, turbidity measurement, filterretardation assays, immunological assays, fluorescent dye bindingassays, protein-staining assays, microscopy, and detection of aggregatesvia ELISA or other binding assay. Structural analysis employing X-raycrystallographic techniques and NMR spectroscopy may also find use. Inone embodiment, stability and/or solubility may be measured bydetermining the amount of protein solution after some defined period oftime. In this assay, the protein may or may not be exposed to someextreme condition, for example elevated temperature, low pH, or thepresence of denaturant. Because function typically requires a stable,soluble, and/or well-folded/structured protein, the aforementionedfunctional and binding assays also provide ways to perform such ameasurement. For example, a solution comprising an immunoglobulin couldbe assayed for its ability to bind target antigen, then exposed toelevated temperature for one or more defined periods of time, thenassayed for antigen binding again. Because unfolded and aggregatedprotein is not expected to be capable of binding antigen, the amount ofactivity remaining provides a measure of the molecule's stability andsolubility.

In one embodiment, molecules may be tested using one or more cell-basedor in vitro assays. For such assays, immunoglobulins, purified orunpurified, are typically added exogenously such that cells are exposedto individual variants or groups of variants belonging to a library.These assays are typically, but not always, based on the biology of theability of the immunoglobulin to bind to the target antigen and mediatesome biochemical event, for example effector functions like cellularlysis, phagocytosis, ligand/receptor binding inhibition, inhibition ofgrowth and/or proliferation, apoptosis and the like. Such assays ofteninvolve monitoring the response of cells to immunoglobulin, for examplecell survival, cell death, cellular phagocytosis, cell lysis, change incellular morphology, or transcriptional activation such as cellularexpression of a natural gene or reporter gene. For example, such assaysmay measure the ability of molecules to elicit ADCC, ADCP, or CDC. Forsome assays additional cells or components, that is in addition to thetarget cells, may need to be added, for example serum complement, oreffector cells such as peripheral blood monocytes (PBMCs), NK cells,macrophages, and the like. Such additional cells may be from anyorganism, e.g., humans, mice, rat, rabbit, and monkey. Crosslinked ormonomeric antibodies may cause apoptosis of certain cell linesexpressing the antibody's target antigen, or they may mediate attack ontarget cells by immune cells which have been added to the assay. Methodsfor monitoring cell death or viability are known in the art, and includethe use of dyes, fluorophores, immunochemical, cytochemical, andradioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferation or activation to be monitored. In oneembodiment, the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer,MA) is used. Alternatively, dead or damaged target cells may bemonitored by measuring the release of one or more natural intracellularproteins, for example lactate dehydrogenase. Transcriptional activationmay also serve as a method for assaying function in cell-based assays.In this case, response may be monitored by assaying for natural genes orproteins which may be upregulated or down-regulated, for example therelease of certain interleukins may be measured, or alternativelyreadout may be via a luciferase or GFP-reporter construct. Cell-basedassays may also involve the measure of morphological changes of cells asa response to the presence of an immunoglobulin. Cell types for suchassays may be prokaryotic or eukaryotic, and a variety of cell linesthat are known in the art may be employed. Alternatively, cell-basedscreens are performed using cells that have been transformed ortransfected with nucleic acids encoding the molecules.

In vitro assays include but are not limited to binding assays, ADCC,CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis ofeffector cells, inhibition of such assays by reduced effector functionantibodies; ranges of activities such as >100× improvement or >100×reduction, blends of receptor activation and the assay outcomes that areexpected from such receptor profiles.

In Vivo Experimentation

The biological properties of the molecules disclosed herein may becharacterized in cell, tissue, and whole organism experiments. As isknown in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. Said animals may be referred to as disease models. Withrespect to the molecules disclosed herein, a particular challenge ariseswhen using animal models to evaluate the potential for in-human efficacyof candidate polypeptides—this is due, at least in part, to the factthat molecules that have a specific effect on the affinity for a humanFc receptor may not have a similar affinity effect with the orthologousanimal receptor. These problems can be further exacerbated by theinevitable ambiguities associated with correct assignment of trueorthologues (Mechetina et al., Immunogenetics, 2002 54:463-468,incorporated entirely by reference), and the fact that some orthologuessimply do not exist in the animal (e.g. humans possess an FcγRIIawhereas mice do not). Therapeutics are often tested in mice, includingbut not limited to mouse strains NZB, NOD, BXSB, MRL/Ipr, K/BxN andtransgenics (including knockins and knockouts). Such mice can developvarious autoimmune conditions that resemble human organ specific,systemic autoimmune or inflammatory disease pathologies such as systemiclupus erythematosus (SLE) and rheumatoid arthritis (RA). For example, animmunoglobulin disclosed herein intended for autoimmune diseases may betested in such mouse models by treating the mice to determine theability of the immunoglobulin to reduce or inhibit the development ofthe disease pathology. Because of the incompatibility between the mouseand human Fcγ receptor system, an alternative approach is to use amurine SCID model in which immune deficient mice are engrafted withhuman PBLs or PBMCs (huPBL-SCID, huPBMC-SCID) providing asemi-functional human immune system with human effector cells and Fcreceptors. In such a model, an antigen challenge (such as tetanustoxoid) activates the B cells to differentiate into plasma cells andsecrete immunoglobulins, thus reconstituting antigen specific humoralimmunity. Therefore, a dual targeting immunoglobulin disclosed hereinthat specifically binds to IgE and FcγRIIb on B cells may be tested toexamine the ability to specifically inhibit B cell differentiation. Suchexperimentation may provide meaningful data for determination of thepotential of said immunoglobulin to be used as a therapeutic. Otherorganisms, e.g., mammals, may also be used for testing. For example,because of their genetic similarity to humans, monkeys can be suitabletherapeutic models, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the immunoglobulins disclosedherein. Tests of the immunoglobulins disclosed herein in humans areultimately required for approval as drugs, and thus of course theseexperiments are contemplated. Thus the immunoglobulins disclosed hereinmay be tested in humans to determine their therapeutic efficacy,toxicity, pharmacokinetics, and/or other clinical properties.

The molecules disclosed herein may confer superior performance onFc-containing therapeutics in animal models or in humans. The receptorbinding profiles of such immunoglobulins, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of suchFc-containing drug. For example, an immunoglobulin with reduced bindingto FcγRIIIa, FcγRI and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an immunoglobulin with reducedbinding to C1q may be selected to limit complement-mediated effectorfunctions. In some contexts, such effector functions are known to havepotential toxic effects. Therefore eliminating them may increase thesafety of the Fc-bearing drug and such improved safety may becharacterized in animal models. In some contexts, such effectorfunctions are known to mediate the desirable therapeutic activity.Therefore enhancing them may increase the activity or potency of theFc-bearing drug and such improved activity or potency may becharacterized in animal models.

In some embodiments, molecules disclosed herein may be assessed forefficacy in clinically relevant animal models of various human diseases.In many cases, relevant models include various transgenic animals forspecific antigens and receptors.

Relevant transgenic models such as those that express human Fc receptors(e.g., CD32b) could be used to evaluate and test immunoglobulins andFc-fusions in their efficacy. The evaluation of molecules by theintroduction of human genes that directly or indirectly mediate effectorfunction in mice or other rodents may enable physiological studies ofefficacy in autoimmune disorders (including SLE, RA and MS). Human Fcreceptors such as FcγRIIb may possess polymorphisms such as that in genepromoter (−343 from G to C) or transmembrane domain of the receptor 187I or T which would further enable the introduction of specific andcombinations of human polymorphisms into rodents. The various studiesinvolving polymorphism-specific FcRs is not limited to this section,however encompasses all discussions and applications of FcRs in generalas specified in throughout this application. Immunoglobulins disclosedherein may confer superior activity on Fc-containing drugs in suchtransgenic models, in particular variants with binding profilesoptimized for human FcγRIIb mediated activity may show superior activityin transgenic CD32b mice. Similar improvements in efficacy in micetransgenic for the other human Fc receptors, e.g. FcγRIIa, FcγRI, etc.,may be observed for molecules with binding profiles optimized for therespective receptors. Mice transgenic for multiple human receptors wouldshow improved activity for immunoglobulins with binding profilesoptimized for the corresponding multiple receptors.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides disclosedherein may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules may mimic—in the animal system—the FcRand/or complement biology of a corresponding candidate humanimmunoglobulin. This mimicry is most likely to be manifested by relativeassociation affinities between specific immunoglobulins and animal vs.human receptors. For example, if one were using a mouse model to assessthe potential in-human efficacy of an Fc variant that has reducedaffinity for the inhibitory human FcγRIIb, an appropriate proxy variantwould have reduced affinity for mouse FcγRII. It should also be notedthat the proxy Fc variants could be created in the context of a human Fcvariant, an animal Fc variant, or both.

In one embodiment, the testing of molecules may include study ofefficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of depletion of specific target cells harboring the targetantigen. Additional primate models include but are not limited to use ofthe rhesus monkey to assess Fc polypeptides in therapeutic studies ofautoimmune, transplantation and cancer.

Toxicity studies are performed to determine antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofiles, or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated, are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabelled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products, also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized, impurities/contaminants have been removed, that the testmaterial is comparable throughout development, that GLP compliance ismaintained.

The pharmacokinetics (PK) of the molecules disclosed herein may bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus and rhesus monkeys. Single orrepeated i.v./s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for half-life (days to weeks) usingplasma concentration and clearance. Volume of distribution at a steadystate and level of systemic absorbance can also be measured. Examples ofsuch parameters of measurement generally include maximum observed plasmaconcentration (Cmax), the time to reach Cmax (Tmax), the area under theplasma concentration-time curve from time 0 to infinity [AUC(0-inf] andapparent elimination half-life (T½). Additional measured parameterscould include compartmental analysis of concentration-time data obtainedfollowing i.v. administration and bioavailability.

The molecules disclosed herein may confer superior pharmacokinetics onFc-containing therapeutics in animal systems or in humans. For example,increased binding to FcRn may increase the half-life and exposure of theFc-containing drug. Alternatively, decreased binding to FcRn maydecrease the half-life and exposure of the Fc-containing drug in caseswhere reduced exposure is favorable such as when such drug hasside-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof molecules disclosed herein. Because molecules of the presentinvention have varying affinities for the array of Fc receptors, furtherscreening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific cells or blocking signaling mechanisms, measuring inhibition ofantigen-specific antibodies etc. The molecules disclosed herein maytarget particular effector cell populations and thereby directFc-containing drugs to induce certain activities to improve potency orto increase penetration into a particularly favorable physiologicalcompartment. For example, neutrophil activity and localization can betargeted by an molecule that targets FcγRIIIb. Such pharmacodynamiceffects may be demonstrated in animal models or in humans.

Use

Once made the molecules as described herein find use in a variety ofmethods. In a preferred embodiment the method includes contacting a cellthat coexpresses IgE and FcγRIIb with a molecule such that both IgE andFcγRIIb are bound by the molecule and the cell is inhibited. By“inhibited” in this context is meant that the molecule is preventing orreducing activation and/or proliferation of IgE+ B cells.

The molecules disclosed herein may find use in a wide range of products.In one embodiment a molecule disclosed herein is a therapeutic, adiagnostic, or a research reagent. The molecules may find use in acomposition that is monoclonal or polyclonal. The molecules disclosedherein may be used for therapeutic purposes. As will be appreciated bythose in the art, the molecules disclosed herein may be used for anytherapeutic purpose that antibodies, and the like may be used for. Themolecules may be administered to a patient to treat disorders includingbut not limited to autoimmune and inflammatory diseases, infectiousdiseases, and cancer.

A “patient” for the purposes disclosed herein includes both humans andother animals, e.g., other mammals. Thus the molecules disclosed hereinhave both human therapy and veterinary applications. The term“treatment” or “treating” as disclosed herein is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor a disease or disorder. Thus, for example, successful administrationof an molecule prior to onset of the disease results in treatment of thedisease. As another example, successful administration of an optimizedmolecule after clinical manifestation of the disease to combat thesymptoms of the disease comprises treatment of the disease. “Treatment”and “treating” also encompasses administration of an optimizedimmunoglobulin after the appearance of the disease in order to eradicatethe disease. Successful administration of an agent after onset and afterclinical symptoms have developed, with possible abatement of clinicalsymptoms and perhaps amelioration of the disease, comprises treatment ofthe disease. Those “in need of treatment” include mammals already havingthe disease or disorder, as well as those prone to having the disease ordisorder, including those in which the disease or disorder is to beprevented.

In one embodiment, a molecule disclosed herein is administered to apatient having a disease involving inappropriate expression of a proteinor other molecule. Within the scope disclosed herein this is meant toinclude diseases and disorders characterized by aberrant proteins, duefor example to alterations in the amount of a protein present, proteinlocalization, posttranslational modification, conformational state, thepresence of a mutant or pathogen protein, etc. Similarly, the disease ordisorder may be characterized by alterations molecules including but notlimited to polysaccharides and gangliosides. An overabundance may be dueto any cause, including but not limited to overexpression at themolecular level, prolonged or accumulated appearance at the site ofaction, or increased activity of a protein relative to normal. Includedwithin this definition are diseases and disorders characterized by areduction of a protein. This reduction may be due to any cause,including but not limited to reduced expression at the molecular level,shortened or reduced appearance at the site of action, mutant forms of aprotein, or decreased activity of a protein relative to normal. Such anoverabundance or reduction of a protein can be measured relative tonormal expression, appearance, or activity of a protein, and saidmeasurement may play an important role in the development and/orclinical testing of the immunoglobulins disclosed herein.

Disclosed herein are novel methods of treating IgE-mediated disorders,e.g., food and environmental allergies and allergic asthma. In preferredembodiments, allergic diseases that may be treated by the products andmethods of the invention include allergic and atopic asthma, atopicdermatitis and eczema, allergic rhinitis, allergic conjunctivitis andrhinoconjunctivitis, allergic encephalomyelitis, allergic rhinitis,allergic vasculitis, and anaphylactic shock. Environmental and foodallergies that may be treated include allergies to dustmite, cockroach,cat and other animals, pollen (including ragweed, Bermuda grass, Russianthistle, oak, rye, and others), molds and fungi (e.g., Alternariaalternata, Aspergillus and others), latex, insect stings (bee, wasp, andothers), penicillin and other drugs, strawberries and other fruits andvegetables, peanuts, soy, and other legumes, walnuts and other treenuts,shellfish and other seafood, milk and other dairy products, wheat andother grains, and eggs. Indeed, any food allergen, aeroallergen,occupational allergen, or other IgE-mediated environmental allergen maybe treated by a therapeutic amount of the products disclosed in thisinvention. For examples of common allergens, see Arbes et al.,Prevalences of positive skin test responses to 10 common allergens inthe US population: Results from the Third National Health and NutritionExamination Survey, Clinical Gastroenterology 116(2), 377-383 (2005).

Also disclosed are diagnostic tests to identify patients who are likelyto show a favorable clinical response to a molecule disclosed herein, orwho are likely to exhibit a significantly better response when treatedwith an molecule disclosed herein versus one or more currently usedtherapeutics. Any of a number of methods for determining FcγRpolymorphisms in humans known in the art may be used. Furthermore, alsodisclosed are prognostic tests performed on clinical samples such asblood and tissue samples. Such tests may assay for activity, regardlessof mechanism. Such information may be used to identify patients forinclusion or exclusion in clinical trials, or to inform decisionsregarding appropriate dosages and treatment regimens. Such informationmay also be used to select a drug that contains a particular moleculethat shows superior activity in such assay.

Formulation

Pharmaceutical compositions are contemplated wherein an moleculedisclosed herein and one or more therapeutically active agents areformulated. Formulations of the molecules disclosed herein are preparedfor storage by mixing said immunoglobulin having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed., 1980, incorporated entirely by reference), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In one embodiment, thepharmaceutical composition that comprises the immunoglobulin disclosedherein may be in a water-soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Some embodiments include atleast one of the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The formulations to be used for in vivo administration maybe sterile. This is readily accomplished by filtration through sterilefiltration membranes or other methods.

The molecules disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing theimmunoglobulin are prepared by methods known in the art. The componentsof the liposome are commonly arranged in a bilayer formation, similar tothe lipid arrangement of biological membranes. Particularly usefulliposomes can be generated by the reverse phase evaporation method witha lipid composition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The molecule and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference. Sustained-releasepreparations may be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymer, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andgamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LupronDepot® (which are injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate),poly-D-(−)-3-hydroxybutyric acid, and ProLease® (commercially availablefrom Alkermes), which is a microsphere-based delivery system composed ofthe desired bioactive molecule incorporated into a matrix ofpoly-DL-lactide-co-glycolide (PLG).

Administration

Administration of the pharmaceutical composition comprising an moleculedisclosed herein, e.g., in the form of a sterile aqueous solution, maybe done in a variety of ways, including, but not limited to orally,subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly. In some instances, for examplefor the treatment of wounds, inflammation, etc., the immunoglobulin maybe directly applied as a solution or spray. As is known in the art, thepharmaceutical composition may be formulated accordingly depending uponthe manner of introduction.

Subcutaneous administration may be used in circumstances where thepatient may self-administer the pharmaceutical composition. Many proteintherapeutics are not sufficiently potent to allow for formulation of atherapeutically effective dose in the maximum acceptable volume forsubcutaneous administration. This problem may be addressed in part bythe use of protein formulations comprising arginine-HCl, histidine, andpolysorbate. Antibodies disclosed herein may be more amenable tosubcutaneous administration due to, for example, increased potency,improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies disclosed herein may also be deliveredusing such methods. For example, administration may be by intravenousinfusion with 0.9% sodium chloride as an infusion vehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Antibodies disclosed herein may be moreamenable to intrapulmonary delivery. FcRn is present in the lung, andmay promote transport from the lung to the bloodstream (e.g. Syntonix WO04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci. 101:9763-8, bothincorporated entirely by reference). Accordingly, antibodies that bindFcRn more effectively in the lung or that are released more efficientlyin the bloodstream may have improved bioavailability followingintrapulmonary administration. Antibodies disclosed herein may also bemore amenable to intrapulmonary administration due to, for example,improved solubility or altered isoelectric point.

Furthermore, molecules disclosed herein may be more amenable to oraldelivery due to, for example, improved stability at gastric pH andincreased resistance to proteolysis. Furthermore, FcRn appears to beexpressed in the intestinal epithelia of adults, so antibodies disclosedherein with improved FcRn interaction profiles may show enhancedbioavailability following oral administration. FcRn mediated transportof antibodies may also occur at other mucus membranes such as those inthe gastrointestinal, respiratory, and genital tracts.

In addition, any of a number of delivery systems are known in the artand may be used to administer the antibodies disclosed herein. Examplesinclude, but are not limited to, encapsulation in liposomes,microparticles, microspheres (e.g., PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamicacid and ethyl-L-glutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the Lupron Depot®, andpoly-D-(−)-3-hydroxybutyric acid. It is also possible to administer anucleic acid encoding an immunoglobulin disclosed herein, for example byretroviral infection, direct injection, or coating with lipids, cellsurface receptors, or other transfection agents. In all cases,controlled release systems may be used to release the immunoglobulin ator close to the desired location of action.

Dosing

The dosing amounts and frequencies of administration are, in oneembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active molecule in theformulation may vary from about 0.1 to 100 weight %. In one embodiment,the concentration of the molecule is in the range of 0.003 to 1.0 molar.In order to treat a patient, a therapeutically effective dose of theimmunoglobulin disclosed herein may be administered. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.0001 to 100 mg/kg of bodyweight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight.In one embodiment, dosages range from 1 to 10 mg/kg.

In some embodiments, only a single dose of the molecule is used. Inother embodiments, multiple doses of the molecule are administered. Theelapsed time between administrations may be less than 1 hour, about 1hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours,about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the molecules disclosed herein are administered inmetronomic dosing regimes, either by continuous infusion or frequentadministration without extended rest periods. Such metronomicadministration may involve dosing at constant intervals without restperiods. Typically such regimens encompass chronic low-dose orcontinuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the molecules disclosed herein and one or moreother prophylactic or therapeutic agents are cyclically administered tothe patient. Cycling therapy involves administration of a first agent atone time, a second agent at a second time, optionally additional agentsat additional times, optionally a rest period, and then repeating thissequence of administration one or more times. The number of cycles istypically from 2-10. Cycling therapy may reduce the development ofresistance to one or more agents, may minimize side effects, or mayimprove treatment efficacy.

Combination Therapies

The molecules disclosed herein may be administered concomitantly withone or more other therapeutic regimens or agents. Additional therapeuticregimes or agents may be used to treat the same disease, to treat anaccompanying complication, or may be used to improve the efficacy orsafety of the immunoglobulin

Particularly preferred co-therapies include those that are approved orare being clinically evaluated for the treatment of IgE-mediateddisorders such as allergies and asthma. In particular, the therapeuticcompositions of the invention may be used in combination withanti-inflammatories such as corticosteroids, and/or brochodilators suchas inhaled β2-agonists, the two major groups of medications. Inhaledcorticosteroids include fluticasone, budesonide, flunisolide,mometasone, triamcinolone, and beclomethasone, whereas oralcorticosteroids include prednisone, methylprednisolone, andprednisolone. Other steroids include glucocorticoids, dexamethasone,cortisone, hydroxycortisone, azulfidineicosanoids such asprostaglandins, thromboxanes, and leukotrienes, as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene.Bronchodilators increase the diameter of the air passages and ease theflow to and from the lungs. Brochodilators that may be combined with thetherapies of the invention include short-acting bronchodilators such asmetaproterenol, ephedrine, terbutaline, and albuterol, and long-actingbronchodilators such as salmeterol, metaproterenol, and theophylline.

The therapies of the invention may be combined with non-steroidalanti-inflammatory drugs (NSAIDs) such as asprin, ibuprofen, celecoxib,diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin,nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, andnabumetone. Co-therapies may include antihistamines such as loratadine,fexofenadine, cetirizine, diphenhydramine, chlorpheniramine maleate,clemastine, and azelastine. Co-therapy may include cromoglycate,cromolyn sodium, and nedrocromil, as well as decongestants, spray ororal, such as oxymetazoline, phenylephrine, and pseudoephedrine. Thetherapies of the invention may be combined with a class ofanti-inflammatories called leukotriene-receptor antagonists such aspranlukast, zafirlukast, and montelukast, and leukotriene-receptorsynthesis-inhibitors such as zileuton.

The therapies of the invention may be combined with otherimmunotherapies, including allergy shots, as well as other antagonistsof IgE or FcεRs. The therapies of the invention may be combined withantagonists of chemokines or cytokines, including but not limited toantibodies and Fc fusions, including but not limited to inhibitors ofchemokines CCR3, CCR4, CCR8, and CRTH2, and CCR5, and inhibitors ofcytokines IL-13, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-15, IL-18,IL-19, IL-21, Class II family of cytokine receptors, IL-22, IL-23,IL-25, IL-27, IL-31, and IL-33. The therapies of the invention may becombined with modulators of adhesion, transcription factors, and/orintracellular signaling. For example, the immunoglobulins of theinvention may be combined with modulators of NF-κb, AP-1, GATA-3, Stat1,Stat-6, c-maf, NFATs, suppressors of cytokine signaling (SOCS),peroxisome proliferator-activated receptors (PPARs), MAP kinase, p38MAPK, JNK, and sphingosine I-phosphate receptors. The therapies of theinvention may be administered with suplatast tolilate, inhibitors ofphosphodiesterase 4 (PDE4), calcium channel blockers, and heparin-likemolecules. Possible co-therapies for the invention are described furtherin detail in Caramori et al., 2008, Journal of Occupational Medicine andToxicology 3-S1-S6.

The therapies of the invention may also be used in conjunction with oneor more antibiotics, anti-fungal agents, or antiviral agents. Theantibodies disclosed herein may also be combined with other therapeuticregimens such as surgery.

EXAMPLES

Examples are provided below are for illustrative purposes only. Theseexamples are not meant to constrain any embodiment disclosed herein toany particular application or theory of operation.

Example 1. Novel Methods for Inhibiting IgE+ FcγRIIb+ Cells

Immunoglobulin IgE is a central initiator and propagator of allergicresponse in affected tissue. IgE binds the high affinity receptor forIgE (FcεRI), a key receptor involved in immediate allergicmanifestations that is expressed on a variety of effector cells,including mast cells, basophils, eosinophils, as well as other celltypes. Cross-linking of FcεRI by immune-complexed IgE-allergen activatesthese cells, releasing chemical mediators such as histamine,prostaglandins, and leukotrienes, which may lead to the development of atype I hypersensitivity reaction. The approved monoclonal antibodyOmalizumab (Xolair) neutralizes IgE by binding to it and blockingengagement with FcεR's. Omalizumab reduces bioactive IgE throughsequestration, attenuating the amount of antigen-specific IgE that canbind to and sensitize tissue mast cells and basophils. Thisneutralization of free circulating IgE, in turn, leads to a decrease insymptoms of allergic diseases. Interestingly, serum IgE levels increaseafter start of therapy because of omalizumab-IgE complex formation andmay remain high up to a year after stopping therapy. Consequently, thisissue may lead to false-negatives on diagnostic tests and therefore IgElevels must be routinely checked.

A novel approach to targeting the IgE pathway involves not only blockingfree circulating IgE from engaging FcεRs on effector cells, buttargeting the source of IgE production. IgE is secreted by IgE-producingplasma cells located in lymph nodes draining the site of antigen entryor locally at the sites of allergic reactions. IgE-producing plasmacells are differentiated from IgE+ B cells. Class switching of B cellsto IgE production is induced by two separate signals, both of which canbe provided by TH2 cells.

There are two forms of immunoglobulins: the secreted and themembrane-anchored form. The membrane-anchored form differs from thesecreted form in that the former has a membrane-anchoring peptideextending from the C terminus of the heavy-chain. Membrane-anchoredimmunoglobulin on B-cells, also referred to as the B cell receptor (BCR)complex, is critical for B-cell functions. It can transduce signals forresting B cells to differentiate into activated lymphoblasts andIg-secreting plasma cells.

Differentiated B cells expressing membrane-anchored IgE, referred tohere as mIgE+ B cells, possess a natural negatively regulating feedbackmechanism—the inhibitory Fc receptor FcγRIIb. FcγRIIb is expressed on avariety of immune cells, including B cells, dendritic cells, monocytes,and macrophages, where it plays a critical role in immune regulation. Inits normal role on B cells, FcγRIIb serves as a feedback mechanism tomodulate B cell activation through the B cell receptor (BCR). Engagementof BCR by immune complexed antigen on mature B cells activates anintracellular signaling cascade, including calcium mobilization, whichleads to cell proliferation and differentiation. However, as IgGantibodies with specificity to the antigen are produced, the associatedimmune complexes (ICs) can crosslink the BCR with FcγRIIb, whereupon theactivation of BCR is inhibited by engagement of FcγRIIb and associatedintracellular signaling pathways that interfere with the downstreampathways of BCR activation. The expression of FcγRIIb on the surface ofmIgE+ B cells, which use mIgE as their BCR, serves as a negativeregulator of these cell types.

A novel strategy for inhibiting IgE-mediated disease, illustrated inFIG. 1, is to inhibit IgE+ B cells (i.e. B cells expressing membraneanchored IgE) by coengaging membrane anchored IgE and the inhibitoryreceptor FcγRIIb. In B cells that have class-switched to express IgE,mIgE serves as the BCR (referred to herein as mIgE BCR). This approachwould potentially mimic the natural biological mechanism of immunecomplex-mediated suppression of B cell activation, thereby preventingdifferentiation into IgE-producing plasma cells. IgE-producing plasmacells reside in the bone marrow and probably have a life span of severalweeks to several months. Since new IgE-secreting plasma cells go throughmIgE-expressing B-cell stages during differentiation, if theirgeneration is abrogated by inhibiting their mIgE+ B cell precursors withthis anti-IgE treatment, the existing plasma cells will die off withinweeks to months, and thus the production of IgE will also graduallyabate. Importantly, inhibition of IgE+ memory B cells, which bear mIgE,would also be inhibited by anti-IgE immunoglobulins that coengageFcγRIIb with high affinity. If this occurs, therapy may have long-termimpact on the fundamental disease.

Example 2. Anti-IgE Antibodies with High Affinity for FcγRIIb

Under physiological conditions, bridging of the BCR with FcγRIIb andsubsequent B cell suppression occurs via immune complexes of IgGs andcognate antigen. The design strategy was to reproduce this effect usinga single crosslinking antibody. Human IgG binds human FcγRIIb with weakaffinity (greater than 100 nM for IgG1), and FcγRIIb-mediated inhibitionoccurs in response to immune-complexed but not monomeric IgG. It wasreasoned that high affinity to this receptor (less than 100 nM) would berequired for maximal inhibition of B cell activation. In order toenhance the inhibitory activity of the anti-IgE antibodies of theinvention, the Fc region was engineered with variants that improvebinding to FcγRIIb. Engineered Fc variants have been described that bindto FcγRIIb with improved affinity relative to native IgG1 (U.S. Ser. No.12/156,183, filed May 30, 2008, entitled “Methods and Compositions forInhibiting CD32b Expressing cells”, herein incorporated expressly byreference).

Variants were originally generated in the context of an antibodytargeting the antigen CD19, a regulatory component of the BCR coreceptorcomplex. The Fv region of this antibody is a humanized and affinitymatured version of antibody 4G7, and is referred to herein as HuAM4G7.The Fv genes for this antibody were subcloned into the mammalianexpression vector pTT5 (National Research Council Canada). Mutations inthe Fc domain were introduced using site-directed mutagenesis(QuikChange, Stratagene, Cedar Creek, Tex.). In addition, control knockout variants with ablated affinity for Fc receptors were generated thatcomprise the substitutions G236R and L328R (G236R/L328R). This variantis referred to as Fc-KO or Fc knockout. Heavy and light chain constructswere cotransfected into HEK293E cells for expression, and antibodieswere purified using protein A affinity chromatography (PierceBiotechnology, Rockford, Ill.).

Recombinant human FcγRIIb protein for binding studies was obtained fromR&D Systems (Minneapolis, Minn.). Genes encoding FcγRIIa and FcγRIIIareceptor proteins were obtained from the Mammalian Gene Collection(ATCC), and subcloned into pTT5 vector (National Research CouncilCanada) containing 6×His tags. Allelic forms of the receptors (H131 andR131 for FcγRIIa and V158 and F158 for FcγRIIIa) were generated usingQuikChange mutagenesis. Vectors encoding the receptors were transfectedinto HEK293T cells, and proteins were purified using nickel affinitychromatography.

Variants were tested for receptor affinity using Biacore technology,also referred to as Biacore herein, a surface plasmon resonance (SPR)based technology for studying biomolecular interactions in real time.SPR measurements were performed using a Biacore 3000 instrument(Biacore, Piscataway, N.J.). A protein NG (Pierce Biotechnology) CM5biosensor chip (Biacore) was generated using a standard primary aminecoupling protocol. All measurements were performed using HBS-EP buffer(10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% vol/vol surfactantP20, Biacore). Antibodies at 20 nM or 50 nM in HBS-EP buffer wereimmobilized on the protein A/G surface and FcγRs were injected. Aftereach cycle, the surface was regenerated by injecting glycine buffer (10mM, pH 1.5). Data were processed by zeroing time and response before theinjection of FcγR and by subtracting appropriate nonspecific signals(response of reference channel and injection of running buffer). Kineticanalyses were performed by global fitting of binding data with a 1:1Langmuir binding model using BIAevaluation software (Biacore).

A representative set of sensorgrams for binding of select variantanti-CD19 antibodies to FcγRIIb is shown in FIG. 2. The affinities ofall variants and WT (native) IgG1 to all of the FcγRs, obtained fromfits of the Biacore binding data, are plotted in FIG. 3 and providednumerically in FIG. 4. Whereas WT IgG1 Fc binds with FcγRIIb with μMaffinity (KD=1.8 μM in FIG. 4), a number of variants, for exampleG236D/S267E, S239D/S267E, and S267E/L328F, have been engineered thatbind the inhibitory receptor more tightly. The S239D/I332E variant, asdescribed in U.S. Ser. No. 11/124,620, also has improved affinity forthe activating receptors FcγRIIa and FcγRIIIa, and therefore is capableof mediated enhanced antibody-dependent cell-mediated cytotoxicity(ADCC) and phagocytosis (ADCP). The G236R/L328R variant, also referredto Fc-knockout or Fc-KO, lacks binding to the Fc receptors, and is usedas a control in the experiments described herein.

Select variants were constructed in antibodies that target IgE. Theheavy and light chain variable regions (VH and VL) of anti-IgEantibodies are provided in FIG. 5. Omalizumab is a humanized antibodythat is currently approved for the treatment of allergic asthma, and ismarketed under the name Xolair. MaE11 is the murine precursor ofOmalizumab. H1L1_MaE11 is a novel humanized version of MaE11. Genesencoding the heavy and light VH and VL domains of these anti-IgEantibodies were synthesized commercially (Blue Heron Biotechnologies).Also synthesized were the variable region VH and VL genes of theanti-respiratory syncytial virus (RSV) antibody motavizumab, used in theexperiments described herein as a negative control. VL genes weresubcloned into the mammalian expression vector pTT5 (NRC-BRI, Canada)encoding the Ckappa constant chain. VH genes were subcloned into thepTT5 vector encoding native IgG1 and variant constant chains. Amino acidsequences of select constant chains are provided in FIG. 6. All DNA wassequenced to confirm the fidelity of the sequences. The amino acidsequences of the full length heavy and light chains of select antibodiesare provided in FIG. 7. As shown in FIG. 9 and discussed in furtherdetail herein, H1L1_MaE11 shows higher affinity to IgE than Omalizumab.

Plasmids containing heavy and light chain genes were co-transfected intoHEK293E cells using lipofectamine (Invitrogen) and grown in FreeStyle293 media (Invitrogen). After 5 days of growth, the antibodies werepurified from the culture supernatant by protein A affinity usingMabSelect resin (GE Healthcare).

Variant and native IgG1 anti-IgE antibodies were tested for binding toIgE and to FcγRIIb using Biacore. DNA encoding the Fc region of IgE,which contains the binding site for the anti-IgE antibodies used, wassynthesized (Blue Heron Biotechnologies) and subcloned into the pTT5vector. IgE Fc was expressed in 293E cells and purified using protein Aas described above. SPR measurements were performed using the proteinA/antibody capture method described above, except that analyte waseither FcγRIIb or the Fc region of IgE. Data acquisition and fitting areas described above. FIG. 8 provides the resulting equilibrium bindingconstants (KDs) obtained from these binding experiments, as well as thefold affinity relative to native IgG1 for binding to FcγRIIb. FIG. 9shows plots of these data. The results confirm the high of affinity ofthe antibodies for IgE, and that the S267E/L328F variant improvesbinding to FcγRIIb over two orders of magnitude, consistent withprevious results.

The use of particular variants, for example S267E/L328F and S239D/1332E,are meant here as proof of concept for the mechanism as describedherein, and are not meant to constrain the invention to their particularuse. The data provided in U.S. Ser. No. 12/156,183 and U.S. Ser. No.11/124,620 indicate that a number of engineered variants, at specific Fcpositions, provide the targeted properties. Substitutions to enhanceFcγR affinity, in particular to FcγRIIb, include: 234, 235, 236, 237,239, 266, 267, 268, 325, 326, 327, 328, and 332. In some embodiments,substitutions are made to at least one or more of the nonlimitingfollowing positions to enhance affinity to FcγRIIb: 235, 236, 239, 266,267, 268, and 328.

Nonlimiting combinations of positions for making substitutions toenhance affinity to FcγRIIb include: 234/239, 234/267, 234/328, 235/236,235/239, 235/267, 235/268, 235/328, 236/239, 236/267, 236/268, 236/328,237/267, 239/267, 239/268, 239/327, 239/328, 239/332, 266/267, 267/268,267/325, 267/327, 267/328, 267/332, 268/327, 268/328, 268/332, 326/328,327/328, and 328/332. In some embodiments, combinations of positions formaking substitutions to enhance affinity to FcγRIIb include, but are notlimited to: 235/267, 236/267, 239/268, 239/267, 267/268, and 267/328.

Substitutions for enhancing affinity to FcγRIIb include: 234D, 234E,234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M,267D, S267E, 268D, 268E, 327D, 327E, L328F, 328W, 328Y, and 332E. Insome embodiments, combination of positions for making substitutions forenhancing affinity to FcγRIIb include, but are not limited to: 235Y,236D, 239D, 266M, S267E, 268D, 268E, L328F, 328W, and 328Y.

Combinations of substitutions for enhancing affinity to FcγRIIb include:L234D/S267E, L234E/S267E, L234F/S267E, L234E/L328F, L234W/S239D,L234W/S239E, L234W/S267E, L234W/L328Y, L235D/S267E, L235D/L328F,L235F/5239D, L235F/S267E, L235F/L328Y, L235Y/G236D, L235Y/S239D,L235Y/S267D, L235Y/S267E, L235Y/H268E, L235Y/L328F, G236D/S239D,G236D/S267E, G236D/H268E, G236D/L328F, G236N/S267E, G237D/S267E,G237N/S267E, S239D/S267D, S239D/S267E, S239D/H268D, S239D/H268E,S239D/A327D, S239D/L328F, S239D/L328W, S239D/L328Y, S239D/I332E,S239E/S267E, V266M/S267E, S267D/H268E, S267E/H268D, S267E/H268E,S267E/N325L, S267E/A327D, S267E/A327E, S267E/L328F, S267E/L328I,S267E/L328Y, S267E/I332E, H268D/A327D, H268D/L328F, H268D/L328W,H268D/L328Y, H268D/I332E, H268E/L328F, H268E/L328Y, A327D/L328Y,L328F/1332E, L328W/1332E, and L328Y/1332E. In some embodiments,combinations of substitutions for enhancing affinity to FcγRIIb include,but are not limited to: L235Y/S267E, G236D/S267E, S239D/H268D,S239D/S267E, S267E/H268D, S267E/H268E, and S267E/L328F.

Example 3. In Vitro Inhibition of IgE+ B Cells by Anti-IgE Antibodieswith High Affinity to FcγRIIb

An enzyme-linked immunosorbent assay (ELISA) was established to detectIgE. Flat bottom plates were prepared by coating with pH 9.4 NaBicarbonate buffer, followed by adherence with anti-IgE captureantibodies at 10 ug/ml overnight in pH 9.4 (0.1 M NaBicarbonate buffer).After overnight, the plate was blocked with 3% BSA/PBS, and serialdilutions of IgE (from a human IgE ELISA kit, Bethyl Laboratories) wasadded 3× to 1 ug/ml. After 3 hours, plates were washed 3× (200 μl) withTTBS, and bound IgE was measured. HRP-conjugated goat polyclonalanti-human IgE antibody (Bethyl Laboratories) was added at (1:5000) for1 hour in 1% BSA/PBS. Samples were washed 3× and IgE was detected withTMB peroxidase substrate (KPL, Inc 50-76-00). Reactions were stoppedwith 50 μl 2N H2SO₄ and read at 450 nm.

FIG. 10 shows capture of IgE with various anti-human IgE antibodies,including a pool of three monoclonal anti-IgE antibodies (MabTech;107/182/101), MaE11_IgG1_G236R/L328R, and Omalizumab_IgG1_G236R/L328R.The data show that the commercial anti-IgE antibody reagent (MabTech),Omalizumab, and its parent chimeric antibody MaE11 are able to captureIgE. In order to use this assay to detect IgE, it was necessary todetermine whether MaE11 and omalizumab antibodies would interfere withIgE capture by the MabTech anti-IgE reagent. The assay was repeated asdescribed above, and concentration of IgE from absorbance was calculatedusing a standard curve. FIG. 11 shows that anti-IgE antibodyomalizumab_G236R/L328R does not compete with the MabTech anti-IgEantibody in the current ELISA protocol.

Fc variant anti-IgE antibodies were tested for their capacity to inhibitIgE+ B cells. Human PBMCs were induced to class switch to IgE producingB cells by adding 5 ng/ml interleukin-4 (IL-4) and 100 ng/ml anti-CD40antibody (clone G28.5 IgG1). The anti-CD40 antibody is an agonist ofCD40, and thus mimics the activity of the co-activator CD40L. Varyingconcentration of anti-IgE antibodies were added, and the samples wereincubated for 12 days. ELISA plates were prepared and blocked asdescribed above, using 5 ug/ml Mabtech anti-IgE as the capture antibody.100 μl of the PBMC samples were added and incubated >3 hours, and thenwashed with TTBS 3× (200 μl). Antibody-HRP conjugated antibody was addedand detected as described above. Absorbance at 450 nm was converted toIgE concentration using a standard curve. The results are shown in FIG.12. Antibodies lacking FcγR binding (G236R/L328R variants) or having nospecificity for IgE (Motavizumab anti-RSV antibody) had no effect on IgEproduction from differentiated B cells. In contrast, variant antibodieswith greater affinity for FcγRIIb inhibited IgE production. These datasuggest that co-engagement of surface IgE and the inhibitory FcγRreceptor FcγRIIb inhibits class-switched B cells of that immunoglobulintype. Inhibition of IgE+ B cells reduces the number of IgE expressingplasma cells, which in turn reduces the amount of IgE detected. Toevaluate the selectivity of this activity for IgE producing B cells,human IgG2 was measured from the same samples using an IgG2 ELISA(Bethyl Laboratories). FIG. 13 shows that IgG2 secretion was notinhibited, indicating that the inhibitory activity of anti-IgEantibodies with high FcγRIIb affinity is selective for IgE+class-switched cells. Repeat of this experiment using variant versionsof the approved anti-IgE antibody Omalizumab showed similar inhibitoryresults by the variant with high FcγRIIb affinity (FIG. 14).

The capacity of anti-IgE antibodies with high FcγRIIb affinity toinhibit IgE production was evaluated in the presence of mIgE BCRstimulation. The above assay was repeated, with class-switching to IgEpromoted by IL-4 and α-CD40 agonist antibody, and in addition the Bcells were activated using either anti-mu or anti-CD79b antibody. Theseantibodies cross-link the BCR, thereby providing a signal similar toimmune-complexed antigen. Anti-mu antibody cross-links membrane-anchoredIgM, and anti-CD79b cross-links CD79b, which is a signaling component ofthe BCR complex. PBMCs were incubated for 14 days with IL-4, α-CD40, andeither anti-CD79b or anti-mu, and IgE was detected as described above.The results for anti-CD79b (FIG. 15) and anti-mu (FIG. 16) show that theanti-IgE antibodies with high affinity for FcγRIIb are capable ofinhibiting IgE production when B cells are stimulated via BCRcross-linking.

An additional strategy for inhibiting IgE+ B cells is to deplete them.This may be carried out using an anti-IgE antibody that is enhanced foreffector function. The variant S239D/I332E increases binding toactivating receptor FcγRIIa and FcγRIIIa (FIG. 3 and FIG. 4), and thusimproves ADCC and ADCP effector functions. The above B cell assay wascarried out using a S239D/I332E variant of the anti-IgE antibodyOmalizumab. PBMCs were incubated for 14 days with IL-4, α-CD40, andeither anti-CD79b (FIG. 17) or anti-mu (FIG. 18), and IgE was detectedas described above. The results (FIGS. 17 and 18) show that anti-IgEantibodies with optimized effector function are able to inhibit IgEproduction from class-switched IgE+ B cells.

Example 4. In Vivo Inhibition of IgE+ B Cells by Anti-IgE Antibodieswith High Affinity to FcγRIIb

The immunoglobulins disclosed herein were assessed using a huPBL-SCIDmouse model as a proxy for therapeutic activity in humans. This studyexamined the capacity of the anti-IgE antibodies described here toinhibit B cell activity and plasma cell development in response to acommon human allergen—dust mite protein Der p 1. In this method, humanperipheral blood leukocytes (PBLs) from a blood donor with allergicresponse to Der p 1 were engrafted to immune-deficient SCID mice andtreated with the native or variant anti-IgE antibodies. The mice werechallenged with an antigen to stimulate an immune response, andproduction of immunoglobulins was measured to examine the course of Bcell development into plasma cells.

Blood donors were screened for allergy to dust mite antigen based on thepresence of anti-IgE antibodies against Der p 1. A donor with positivereactivity was leukapheresed to obtained peripheral blood mononuclearcells (PBMCs). The protocol for the study is provided in FIG. 20. Oneday prior to PBMC injection, mice were given intraperitoneal (i.p.)injections with 100 μl of anti-asialo GM antibody (Wako, Richmond, Va.)to deplete murine natural killer (NK) cells. The next day, mice wereinjected i.p. with 3×10⁷ PBLs in a 0.5 ml volume. After PBMC injection,mice were assigned to 5 different groups of mice with 7 mice in eachgroup. On day 7 post PBMC injection, blood was collected from all micevia retro-orbital sinus/plexus (OSP) puncture for determination of humanIgG and IgE levels by ELISA (ZeptoMetrix, Buffalo, N.Y.). Two days later(day 9), mice were injected i.p. with 10 mg/kg antibody or PBS. On day11, mice were injected i.p. with 15 ug dustmite antigen Der p 1 (LoToxNatural Der p 1, Indoor Biotechnologies, Charlottesville, Va.). On day23 (12 days post antigen vaccination), blood was collected from all micefor determination of human IgG and IgE antibodies. On the same day, micereceived a second injection i.p. with 10 mg/kg antibody or PBS. Two dayslater (day 25), mice received a boost vaccination i.p. of 10 ug dustmiteantigen Der p 1. On day 37 (12 days post antigen boost), blood wascollected by OSP for human immunoglobulin determination. Human IgG andIgE concentrations were measured using ELISA methods similar to thosedescribed above.

The results are shown in FIGS. 20 and 21 for serum IgG and IgE levelsrespectively. Before the allergen challenge, the levels of human IgG andIgE antibodies were low in all the groups. After Der p 1 immunization,all groups showed high levels of human IgG, indicating a robust immuneresponse by engrafted human B cells to either the vaccinated Der p 1antigen or endogenous mouse antigens. In contrast to IgG response, thetreatment groups differed significantly in their production of IgEantibodies. Omalizumab and the IgG1 version of H1 L1 MaE11 wereequivalent to vehicle in their capacity to inhibit production of humanIgE. However the FcγRIIb-enhanced (IIbE, S267E/L328F) version of H1 L1MaE11 showed no detectable levels of human IgE. The Fc-KO (variantG236R/L328R) version of H1L1 MaE11, which lacks binding to all FcγRs,showed an enhancement in human IgE production. This is possibly due toits ability to cross-link human mIgE and thus activate IgE+ B cells, yetits complete lack of FcγRIIb inhibitory or FcγRIIa/IIIa cytotoxicactivities such as those possessed by the IgG1 and IIbE versions of theantibody. These in vivo data show that anti-IgE antibodies with highaffinity for FcγRIIb are capable of inhibiting human IgE+ B cellactivation and immunoglobulin secreting plasma cell differentiation, andthus support the potential of the immunoglobulins disclosed herein fortreating IgE-mediated disorders.

Example 5. Comparative PK/PD Model of XmAb7195 Vs. Omalizumab Effect onFree and Total IgE in Chimpanzees

XmAb7195 (anti-human IgE, S267E/L328F) was evaluated for itspharmacokinetics and pharmacodynamics (free and total IgE) inchimpanzees following a single intravenous dose of 5 mg/kg. Chimpanzeesand humans have similar FcγRIIb structure at the critical binding region(Arginine at position 131, or 131-R), in contrast to macaques which donot have the relevant contact amino acid. The comparator antibody inthis study was commercially available omalizumab (Xolair®. Genentech,USA), an anti-human IgE antibody with a wild type human IgG1 Fc domain.

The purpose of this study was two-fold. The first objective was toevaluate the pharmacokinetic behavior of XmAb7195 in chimpanzees.Sequence differences among primate species lead to significantdifferences in receptor affinities for XmAb7195. The receptor-mediatedclearance of XmAb7195 may involve Fc-gamma receptors type II (a and b).PK experiments in other non-human primates have been performed, but maynot be predictive since macaques do not have arginine at position 131 ofthe Fc gamma type II receptors. PK in chimpanzees, which have theappropriate genotype, may be more predictive of the PK/PD profileexpected in human clinical studies. The second purpose of the study wasto evaluate the pharmacodynamic effect of a single dose of XmAb7195 onthe sequestration, production, and clearance of IgE. In each of theseobjectives, we used omalizumab as a comparator molecule in order toevaluate the effect that the engineered Fc had on PK/PD parameters.

The results of free drug concentrations as a function of time arepresented in FIG. 22A. Notably, XmAb7195 has a shorter half-life ofapproximately 2 days compared to the approximately 11 days observed foromalizumab. Analysis of free and total IgE was undertaken on serumsamples at each of the PK time points. Free IgE levels exhibited a rapiddrop immediately after dosing. The nadir of free IgE concentrations forthe omalizumab-treated chimps averaged approximately 50 ng/ml at onehour post dosing. XmAb7195 caused a more significant reduction of freeIgE, reaching levels below the lower limit of quantitation (LLOQ) of 4ng/ml almost immediately after dosing and remaining below the LLOQ up today 10. (FIG. 22B) Omalizumab increased total IgE for a period of weeks,similar to its observed effects in humans. XmAb7195 caused a rapiddisappearance of total IgE—reaching the LLOQ within 1 hour post-dosingand lasting for 10 days—followed by a gradual return to baseline levelsover a period of weeks. FIG. 22C shows group mean total IgE levelsversus time for chimpanzees treated with omalizumab or XmAb7195(anti-IgE, S267E/L328F). The lower limit of quantification was 0.2μg/ml.

Example 6. PK/PD of Anti-IgE Antibodies in Human FcγRIIb Transgenic Mice

Several anti-mouse IgE antibodies with different FcγRIIb-enhancing Fcsubstitutions were produced for comparison of their ability to modulatetotal IgE concentrations in vivo. The first, XENP8253, comprises theR1E4 Fv domain (anti-mouse IgE) and an Fc domain containing theS267E/L328F substitutions. The second, XENP8252, is a surrogate foromalizumab, comprising the R1E4 Fv domain with a native human IgG1backbone. Additional Fc variants—S267E, G236D/S267E, and G236N/S267Ewere also characterized to examine the relationship between humanFcγRIIb affinity and pharmacokinetics and pharmacodynamics.

A single 2 mg/kg dose of all anti-IgE antibodies sequestered serum IgEand reduced free IgE serum levels by several orders of magnitude withinhours of the treatment. Their effect on total IgE, however, was verydifferent. The omalizumab surrogate (XENP8252), which contains anunmodified IgG1 Fc domain, had no discernible effect on total IgErelative to the PBS control. In contrast, the high FcγRIIb affinityvariant S267E/L328F reduced total IgE within hours, and caused sustainedreduction of total IgE relative to both the PBS group and theXENP8252-treated mice. The extremely rapid onset of the total IgEreduction in this model system indicates (without being bound by theory)that anti-IgE antibodies with enhanced affinity for FcγRIIb increase therate of drug:IgE complex clearance. This hypothesis is consistent withthe observation that the S267E/L328F (IIbE) variant has reducedhalf-life (approximately 2.5 days) relative to the IgG1 antibody(XENP8252) (approximately 11 days). (FIG. 23). FIG. 24 shows serum totalIgE concentration as a function of time in the human FcγRIIb transgenicmice treated with anti-mouse IgE antibodies. The lower limit ofquantification of this IgE assay was 13 ng/ml.

The additional variant antibodies—S267E, G236D/S267E, and G236N/S267E,with FcγRIIb affinities intermediate between IgG1 and S267E/L328, haveintermediate half-lives and intermediate effects on total IgE, revealinga direct relationship between FcγRIIb affinity and clearance rates ofeither antibody alone or antibody complexed with antigen. (See FIG. 25).

Example 7. Antibody:IgE Complex Internalization by Liver SinusoidalEndothelial Cells (LSEC) from FcγRIIb Transgenic Mice

A hypothesis tested was that much of the in vivo accelerated clearanceof antibody and antibody:IgE complexes is mediated by liver sinusoidalendothelial cells (LSEC). Anderson and colleagues (Ganesan et al., JImmunol 2012) published a study demonstrating that three quarters ofmouse FcγRIIb is expressed in the liver, with 90% of it being expressedin LSEC. Moreover, the authors demonstrated that clearance ofradiolabeled small immune complexes (SIC) is significantly impaired inan FcγRIIb knockout strain compared to wild-type mice.

An LSEC enrichment protocol was adopted from Katz et al, (Katz et al.,(2004) J Immunol “Liver sinusoidal endothelial cells are insufficient toactivate T cells,” 173(1): 230-235) for non-parenchymal cell isolation.Briefly, mouse liver was infused with 1 ml of 1% (w/v) collagenase D in1×HBSS using syringe and needle. Then, the liver was quickly minced in20 ml of 1% collagenase D and incubated at 37° C. for 20 minutes withstirring to keep the cells in suspension. The cell suspension was passedthrough a 100 μm cell strainer filter mesh and spun 3× at low speed(30×g, 10 minutes) to remove the bulk of parenchymal hepatocytes. Thefinal enriched non-parenchymal liver cell pellet (300×g, 10 minutes) waswashed twice with PBS (300×g, 10 minutes) and used in internalizationassays.

FITC conjugated anti-IgE antibodies plus human IgE pre-formed IC wereincubated with enriched LSEC's for 60 minutes at 37° C., washed with lowpH “Acid” wash buffer (glycine, NaCl, pH=2.7) and stained withanti-CD146 and anti-CD45 antibodies. The internalized signal (FITC MFI)was quantified from CD146+CD45low LSEC's and MESF normalized values areplotted. As shown in FIG. 26, IgE complexes formed with the anti-IgEantibody containing the high IIb affinity variant S267E/L328 internalizeinto LSEC more substantially than either anti-IgE IgG1 or anti-IgE withFc knockout substitutions (G236R/L328R). Variants with intermediate IIbaffinity—S267E, G236D/S267E, and G236N/S267E—displayed intermediateinternalization corresponding with their relative affinities.

Example 8. Whole Body Imaging Study of IgE Biodistribution

A study was designed to evaluate the biodistribution and specifichepatic uptake of XmAb7195 (anti-IgE-S267E/L328F) as compared to salinecontrols and control antibody XENP6728 (anti-IgE-IgG1), whenadministered intravenously together with ⁸⁹Zr-IgE to female FcγRIIbtransgenic mice.

Treatment began on Day 0. Animals were first injected intravenously with⁸⁹Zr-labeled IgE in the range of 0.10 to 0.13 mCi. This was immediatelyfollowed by an intravenous injection of saline, 10 mg/kg XmAb7195, or 10mg/kg XENP6728.

Animals were induced with 3.0% isoflurane in air (2.0 L/min) and weremaintained during imaging procedures at 1.0-2.0% isoflurane in air (2.0L/min). Animals were positioned inside the Siemens Inveon PET ring, andthe PET acquisition was initiated and data was acquired continuously for2 hours for the first acquisition.

Administration of XmAb7195 was associated with significantly greater,more rapid and more sustained accumulation of ⁸⁹Zr-IgE in the liver,compared with saline and compared with the IgG1 analog XENP6728.Correspondingly, administration of XmAb7195 was associated withsignificantly reduced accumulation of ⁸⁹Zr-IgE in the heart, which isrepresentative of the amount of labeled IgE in bulk circulation. (FIG.27).

Example 9. CR2-Fc Fusions with High Affinity for FcγRIIb

Soluble CRs and CR-Fc fusions have been described for therapeuticpurposes. These include CR1, CR2-Fc (U.S. Pat. No. 6,458,360), CR2-fH(CR2-factor H), and others. However, while these approaches generallyblock interaction of C3-tagged ICs with their associated receptors, theydo not necessarily remove the immune complexes from circulation. Most ofthe complement receptors and regulatory proteins are composed of one ormore so-called short complement repeat (SCR) domains, also calledcomplement control protein (CCP) modules or Sushi domains. Typically,only a subset of the domains is involved in direct recognition of theassociated complement fragment ligand. For example, it has beendemonstrated that only the first two SCRs of CR2 are essential for C3dbinding. The SCR domains are stable and well-behaved, making themsuitable for use in the development of therapeutic proteins.

Genes encoding the first two (SCR1-2) or first four (SCR1-4) SCR domainsfrom human and mouse were synthesized commercially (Blue HeronBiotechnologies). Genes were subcloned into the mammalian expressionvector pTT5 (NRC-BRI, Canada) encoding the human IgG1 Fc (hinge-CH2-CH3domains). The SCR domains were also subcloned into pTT5 vectors encodingvariant IgG1 Fc domains containing S267E, G236D/L328F, G236N/L328F,S267E/L328F (high FcRIIb binding), or G236R/L328R (ablated FcR bindingor Fc knockout; also shown as FcKO) substitutions. All DNA was sequencedto confirm the fidelity of the sequences. Amino acid sequences of selectCR2-Fc variants are provided in FIG. 40. Plasmids containing heavy andlight chain genes were co-transfected into HEK293E cells usinglipofectamine (Invitrogen) and grown in FreeStyle 293 media(Invitrogen). After 5 days of growth, the antibodies were purified fromthe culture supernatant by protein A affinity using MabSelect resin (GEHealthcare).

SPR measurements were performed using a Biacore 3000 instrument(Biacore, Piscataway, N.J.). A protein A (Pierce Biotechnology) CM5biosensor chip (Biacore) was generated using a standard primary aminecoupling protocol. All measurements were performed using HBS-EP buffer(10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% vol/vol surfactantP20, Biacore). CR2-Fc variants at 25 nM in HBS-EP buffer wereimmobilized on the protein A surface and then 100 nM purified human C3d(Alpha Diagnostic cat# C3D18-N-25) was injected. After each cycle, thesurface was regenerated by injecting glycine buffer (10 mM, pH 1.5).Data were processed by zeroing time and response before the injection ofC3d and by subtracting appropriate nonspecific signals (response ofreference channel and injection of running buffer). Kinetic analyseswere performed by global fitting of binding data with a 1:1 Langmuirbinding model using BIAevaluation software (Biacore). Example bindingcurves are shown in FIG. 39A.

Binding of CR2-Fc constructs to recombinant C3d-Fc was evaluated usingELISA. XENP12561 or XENP12562 (hSCR1-2 or hSCR1-4 Fc fusion) were coatedto plates followed by adding varying concentrations of XENP12704, whichis an anti-IgE antibody containing human C3d fused to the C-terminus ofCkappa. Anti-C1q antibody and no plate coating were used as controls.Plates were incubated overnight at 4° C. and ananti-IgG-F(ab′)2-specific-HRP antibody was used for ELISA detection.Results are shown in FIG. 39B. XENP12561 and XENP12562 showed clearbinding to recombinant C3d, with XENP12562 (containing hSCR1-4 domains)showing slightly stronger binding. A similar ELISA format was also usedto evaluate the binding of CR2-Fc constructs to C3d-tagged immunecomplexes (IC) present in normal and rheumatoid arthritis (RA) patientsera. RA patients have autoimmune antibodies present and their sera areexpected to contain a higher amount of C3d-tagged IC of these antibodiescompared to normal subjects. XENP12561 or XENP12562 (hSCR1-2 or hSCR1-4Fc fusion) were coated to plates followed by adding varyingconcentrations of normal or RA patient sera. Plates were incubatedovernight at 4° C. and an anti-IgG-F(ab′)2-specific-HRP antibody wasused for ELISA detection. Results are shown in FIG. 39C. XENP12561 andXENP12562 showed strong binding to c3d-tagged IC present in both normaland RA patient sera. These results show that there is approximately100-fold higher amount of IC in RA patient sera compared to normal sera.

Example 10. Design of Fc-Containing oxLDL-Binding Proteins with EnhancedFcγRIIb Affinity

OxLDL is bound naturally by scavenger receptors such as LOX-1 (alsoknown as OLR1) and CD36. Amino acid sequences for human and mouseversions of these receptors are listed in FIG. 43. LOX-1 and CD36 Fcfusions can be designed (XENP13516, XENP13517, XENP13518, sequences arelisted in FIG. 44A-B). An Fc region is desirable to increase serumhalf-life, stability, and expression yields, while also serving as ascaffold for the inclusion of Fc variants for enhancing FcγRIIbaffinity. Also, monoclonal antibodies that bind oxLDL are known in theart (XENP13514 and XENP13515, sequences are listed in FIG. 44A-B).

Plasmids containing appropriate genes were transfected or co-transfectedinto HEK293E cells using lipofectamine (Invitrogen) and grown inFreeStyle 293 media (Invitrogen). After 5 days of growth, the proteinswere purified from the culture supernatant by protein A affinity usingMabSelect resin (GE Healthcare). The resulting proteins were examined bysize-exclusion chromatography (see FIG. 45).

Based on these results, amino acid sequences were designed forFc-containing oxLDL-binding proteins containing the S267E/L328F Fcvariant that confers enhanced FcγRIIb affinity (FIG. 46). Alternative Fcvariants with various levels of FcγRIIb affinity can also be used asmentioned above, e.g., G236N/S236E, G236D/S267E, and S267E. Thesevariants allow for the rapid clearance of oxLDL from the blood.

The EO6 antibody can be humanized to reduce its immunogenicity as atherapeutic in humans. Sequences of humanized variable regions derivedfrom the EO6 parental sequence can be found in FIG. 47.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments have been described above for purposes ofillustration, it will be appreciated by those skilled in the art thatnumerous variations of the details may be made without departing fromthe invention as described in the appended claims.

We claim:
 1. A method of treating atherosclerosis in a patient byrapidly lowering serum concentration of oxidized low-density lipoprotein(oxLDL) in said patient, said method comprising: a) administering arapid clearance molecule comprising: i) a domain that binds said oxLDL;and ii) a variant human IgG Fc domain comprising an amino acidsubstitution as compared to a parent human IgG Fc domain, wherein saidvariant Fc domain binds FcγRIIb with increased affinity as compared tosaid parent Fc domain; wherein said rapid clearance molecule binds tosaid oxLDL to form a molecule-oxLDL complex and said complex is clearedat least two fold faster than oxLDL alone.
 2. A method according toclaim 1, wherein said variant Fc domain comprises amino acidsubstitutions selected from the group consisting of S267E, S267D, L328F,P238D, S267E/L328F, G236N/S267E, and G236D/S267E, wherein numbering isaccording to EU index as in Kabat.
 3. A method according to claim 1,wherein said rapid clearance molecule is an antibody or an Fc fusionprotein.
 4. A method according to claim 3, wherein said rapid clearancemolecule is an anti-oxLDL antibody.
 5. A method according to claim 3,wherein said rapid clearance molecule is a LOX-1 Fc fusion protein.
 6. Amethod according to claim 3, wherein said rapid clearance molecule is aCD36 Fc fusion protein.
 7. A method according to claim 1, wherein saidvariant Fc domain comprises amino acid substitutions selected from thegroup consisting of: N434A, N434S, M428L, V308F, V259I, M428L/N434S,V259I/V308F, Y436I/M428L, Y436I or V/N434S, Y436V/M428L, M252Y,M252Y/S254T/T256E and V259I/V308F/M428L, wherein numbering is accordingto EU index as in Kabat.
 8. A method according to claim 1, wherein saidvariant Fc domain is a variant of a parent human IgG1 Fc domain.